Substrate-selective co-fermentation process

ABSTRACT

Biological method for conversion of a sugar-containing organic material into a desired biochemical product. Use of a plurality of substrate-selective cells allows different sugars in a complex mixture to be consumed concurrently and independently. The method can be readily extended to remove inhibitory compounds from hydrolysate.

This application is a continuation-in-part of U.S. Ser. No. 12/587,466,filed Oct. 7, 2009, which is a continuation-in-part of InternationalApplication No. PCT/US2008/004577, filed Apr. 9, 2008, which claims thebenefit of U.S. Provisional Application Ser. No. 60/922,473, filed Apr.9, 2007, and U.S. Provisional Application Ser. No. 61/004,356, filedNov. 27, 2007, each of which is incorporated by reference herein.

BACKGROUND

Over the last ten years many reports have provided compelling evidencefor a competitively priced bio-based products industry that will replacemuch of the petrochemical industry. In order to reduce U.S. reliance onforeign oil, there is an increasing interest in generating commodity andfine chemicals from widely available U.S. renewable resources throughfermentation. In the last five years, billions of dollars have beeninvested in commercializing the microbial production of severalchemicals, including lactic acid, 1,3-propanediol and 3-hydroxypropanoicacid. In concert with this increased interest in crop-derivedbiochemicals, molecular biology has become a standard tool whichengineers and applied scientists use routinely to aid the rational andtargeted alteration of metabolism.

The efficient use of agricultural biomass for the production of anybiochemical, however, is problematic. Technical challenges to beovercome in order for bio-based industrial products to becost-competitive include finding new technology and reducing the cost oftechnology for converting biomass into desired bio-based industrialproducts. Research resulting in cost-effective technology to overcomethe recalcitrance of cellulosic biomass would allow biorefineries toproduce fuels and bulk chemicals on a very large scale.

The untapped sources of biomass are largely lignocellulosic in nature.One promising use of lignocellulose for liquid fuel is in the microbialproduction of ethanol. Unfortunately, when broken down intoconstituents, a very complex mixture remains. This mixture containssugars which individually but not collectively are suitable forfermentation, and the mixture also contains inhibitors.

Because the unit value of chemical products derived from biomass (e.g.,ethanol) is generally low while the potential market is large, theeconomic viability of such processes depends on the yield andproductivity. Yield is the quantity of product formed per mass ofmaterial input, while productivity is the rate at which the product isgenerated. Achieving high yield demands that all biomass components beconverted, while high productivity requires that the complex conversionsbe accomplished quickly.

The conversion of lignocellulosic biomass to any useful fermentationproduct follows a series of general process operations which includeidentifying the biomass, harvesting it, various separation/extractionsteps, pretreatments, conversion and subsequent purification steps.Although the particular substrate and chemical product determine thedetails of each operation, the complex structure of biomass invariablynecessitates that its components be broken down by variousdepolymerization strategies.

Fermentation is the biological process in which sugar substrates, suchas glucose and xylose, are converted into fermentation products, such asethanol. While the term fermentation is usually reserved for anaerobicprocesses, analogous microbial processes similarly convert sugarsubstrates into products under a controlled aerobic environment or underconditions of partial oxygenation. One major limitation is that,regardless of a microorganism's ability to metabolize multiplesubstrates, a single substrate persistently remains the preferredsubstrate and the consumption of the sugars is asynchronous. Invariably,a multitude of substrates remains long after the preferred substrate hasbeen metabolized. Numerous attempts to engineer microorganisms capableof equally metabolizing more than one substrate have been made (Dien etal., 2002, J. Industr. Micro. 29:221; Sedlak et al., 2003, Enzyme Micro.Technol. 33:19; Chandrakant and Bisaria, 2000, Appl. Micro. Biotechnol.53:301; Kuyper et al., 2005, FEMS Yeast Res. 5:925; Zhang et al., 1995,Science 267:240). However, none has prevented the polyauxic behavior oflinearly metabolizing one sugar at a time when metabolizing sugarmixtures (i.e., first glucose consumption, then xylose, etc.).

A single microorganism alone is unable to convert multiple sugarssimultaneously. Instead, any given microorganism has a complexregulatory network which forces sugars to be metabolized sequentially.This sequential nature invariably reduces the overall rate of afermentation process to generate the desired product.

Hydrolysis of the lignocellulosic biomass releases variety of sugars,including hexoses (e.g., glucose, mannose), pentoses (e.g., xylose,arabinose), and oligosaccharides, that are released by the hydrolysis oflignocellulosic biomass, and no single microorganism is capable offermenting all these sugars. Many ethanologenic microbes, includingyeast, prefer to use glucose as a substrate. Even when yeast cells aremodified genetically to use xylose, they ferment all glucose beforeswitching to the much slower xylose fermentation. Conversion rates canvary greatly depending on such factors as the type of sugar substratebeing fermented, environmental conditions (e.g., pH, temperature), andthe concentrations of certain products from other metabolic pathways.

The fraction of pentose sugars which compose biomass can significant;for example, 12% pentose sugars have been reported for Pinus spp. and26% for Populus spp. (Saddler and Mackie, 1990, Biomass 22:293).Acid/enzymatic hydrolysis of agricultural materials also generates asignificant fraction of pentoses. For example, hydrolysis of peanuthulls results in a mixture containing 44% pentoses (Chandrakant andBisaria, 2000, Appl. Micro. Biotechnol. 53:301). In order to achievehigh yields and productivities, both pentose and hexose fractions mustbe fully and efficiently utilized. Complicating the matter is the factthat hydrolysis also leads to the formation of acetic acid, which is aknown inhibitor to any of the microorganisms that might be used toferment these sugars into products such as ethanol.

The efficient conversion of both pentoses and hexoses is a significanthurdle to the economic utilization of biomass hydrolysates for thegeneration of any fermentation product. The central problem is thateither the microorganism used to metabolize the sugars in the mixtureconsumes the sugar constituents sequentially (e.g., first glucose andthen xylose) or the organism is unable to utilize the pentose at all (asis the case with the yeast, Saccharomyces cerevisiae). In a recentcomprehensive review Zaldivar et al. succinctly conclude “the lack of amicroorganism able to ferment efficiently all sugars released byhydrolysis from lignocellulosic materials has been one of the mainfactors preventing utilization of lignocellulose” (Zaldivar et al.,2001, Appl. Microbiol. Biotechnol. 56:17).

Current strategies have focused on the development of a single organismengineered to metabolize both hexoses and pentoses, a single organismthat “can do it all.” For example, the common yeast Saccharomycescerevisiae, the most widely used organism for ethanol production fromstarch-based crops, has been genetically modified to metabolize xyloseas well as its native substrate glucose. Genes encoding xylosereductase, xylitol dehydrogenase and xylulokinase fused to glycolyticpromoters have been successfully integrated into the yeast chromosome(Ho et al., 1998, Appl. Env. Micro. 64:1852.; Sedlak et al., 2003,Enzyme Micro. Technol. 33:19.). In another study, S. cerevisiaegenetically engineered to contain genes to metabolize xylose stillconsumed less than 25% of the xylose when glucose was depleted (Sedlaket al., 2003, Enzyme Micro. Technol. 33:19.). Even when xylose isomeraseactivity was added to S. cerevisiae to convert xylose to glucoseextracellularly, 75% of the xylose still remained after the glucose wascompletely consumed (Chandrakant and Bisaria, 2000, Appl. Micro.Biotechnol. 53:301.).

Bacteria are also frequently used for fermentation processes, but areunable to efficiently metabolize sugar mixtures. In many bacteria, themetabolism of glucose prevents efficient xylose consumption and as aresult many researchers have attempted to improve the efficiency ofxylose consumption. Introducing a mutation into the ptsG gene ofEscherichia coli can reduce glucose-mediated repression of xyloseconsumption (Dien et al., 2002, J. Industr. Micro. 29:221.). Forexample, in batch culture with the ethanologenic E. coli strain K011grown on hemicellulose hydrolysate, for example, only 11% of the xylosewas consumed after 24 h, while 80% of the glucose was consumed (Barbosaet al., 1992, Appl. Env. Micro. 58:1382.). Removal of the ptsG improvedxylose consumption in the presence of glucose, but still 40% of thexylose remained when the glucose is depleted (Dien et al., 2002, J.Industr. Micro. 29:221.).

Approaches using “evolutionary engineering” have also significantlyimproved the rate of xylose consumption (Kuyper et al., 2005, FEMS YeastRes. 5:925), but have not prevented the diauxic behavior when usingsugar mixtures (i.e., first glucose consumption, then xylose). Likewise,introduction of genes involved in the xylose metabolism pathway intoZymomonas mobilis does not prevent it from consuming xylose much moreslowly than glucose (Zhang et al., 1995, Science 267:240). Because bothsugars are not consumed effectively in any of these single-organismprocesses, the productivity of the process is suboptimal.

Strategies which require a single organism to convert xylose and glucosesuffer from several limitations. First, as noted above, the consumptionof the sugars is asynchronous. Despite the presence of the geneticapparatus to consume both sugars, glucose remains the preferredsubstrate, and xylose invariably remains when glucose has been consumed.This asynchronicity particularly influences a single microorganism'sability to cope well with a real hydrolysate having a temporally varyingconcentration of each sugar. Faced with a time-varying stream, yet usinga single organism which has a limited ability to adjust its ratio ofglucose and xylose consumption rates, the process will invariably leadto one of the sugars not being effectively consumed. It is not currentlypossible for one organism to “adjust” its rate of consumption to twosubstrates in order to match the concentrations encountered in a realprocess.

A second shortcoming is that a single microorganism strain, which hasbeen engineered to consume both glucose and xylose, tends to beunstable. A study using a chemostat demonstrated that the presence ofboth sugars caused an increase over time in the by-product (andinhibitor) acetic acid, which ultimately led to a 20% decrease inethanol yield (Dumsday et al., 1999, J. Indust. Micro. Biotechnol.23:701).

This highlights another complication: hydrolysis of real biomasstypically generates compounds which inhibit the subsequent conversion ofsugars by fermentation. For example, xylose is acetylated inlignocellulose (Timmel, 1967, Wood Sci. Technol. 1:45; Chesson et al.,1993, J. Sci. Food Agric. 34:1330), and therefore acetic acid is anunavoidable product of hemicellulose depolymerization. Although theinhibitory effect of acetic acid depends on the strain and process, anacetic acid concentration of only 0.08% has been demonstrated to inhibita subsequent fermentation to generate ethanol (van Zyl et al., 1991,Enzyme Micro. Technol. 13:82). Generally acetic acid inhibits xyloseconversion more than it affects glucose conversion. In S. cerevisiaeengineered to metabolize xylose, for example, acetic acid reducedethanol yield from xylose by 50% (Helle et al., 2003, Enzyme MicrobialTechnol. 33:786). Acetic acid itself, and not merely the pH, causes theinhibition. Therefore, base neutralization traditionally applied to acidtreated lignocellulosic hydrolysates does not eliminate the inhibitoryaffects of acetic acid. Previous approaches to convert this mixture ofsugars and inhibitors have not been able to achieve the rate ofconversion necessary to make the process economically viable. A novelapproach that successfully overcomes the inability to convert a mixtureof sugars and inhibitors and increases the yields of fermentationproduct per amount of biomass would represent a significant and longawaited advance in the field.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for producing abiochemical that includes concurrently contacting an organic material,preferably an organic material that contains a mixture of sugars, suchas a lignocellulosic hydrolysate, with a plurality of sugar-selectivecells under conditions to allow the plurality of sugar-selective cellsto produce the biochemical. The method optionally includes theadditional steps of hydrolyzing a biomass, such as a lignocellulosicbiomass, to produce the hydrolyzed biomass, such as a hydrolyzedlignocellusosic biomass, and/or purifying the resulting biochemicalproduct. Exemplary biochemicals that can be produced using the method ofthe invention include ethanol, butanol, succinate, lactate, fumarate,pyruvate, butyric acid and acetone.

The plurality of sugar-selective cells may include at least onehexose-selective cell and at least one pentose-selective cell.Preferably, the plurality of sugar-selective cells includes first andsecond sugar-selective cells. The first sugar-selective cell metabolizesa first sugar that cannot be metabolized by the second sugar-selectivecell. The second sugar-selective cell metabolizes a second sugar thatcannot be metabolized by the first sugar-selective cell. These first andsecond sugars are different, and are preferably selected from the groupconsisting of glucose, xylose, arabinose, galactose, or mannose. Morepreferably, at least one of the sugar-selective cells cannot metabolizeat least of those two sugars, more preferably at least three of thosesugars. In an even more preferred embodiment, at least one of thesugar-selective cells can metabolize only one sugar that isindependently selected from the group consisting of glucose, xylose,arabinose, galactose, and mannose.

The sugar selective cells are preferably single-celled microorganisms,such as yeast or bacteria. Cells suitable for use in the method includecells of Escherichia coli, Zymomonas mobilis, Corynebacteriumglutamicum, Lactic Acid Bacteria, Saccharomyces cerevisiae, Pichiastipitis and Ambrosiozyma monospora. In a preferred embodiment, theplurality of cells includes a first E. coli cell wherein at least onegene involved in glucose metabolism has been modified or deleted so asto inhibit or prevent the first E. coli cell from consuming glucose; anda second E. coli cell wherein at least one gene involved in xylosemetabolism has been modified or deleted so as to inhibit or prevent thesecond E. coli cell from consuming xylose.

Optionally, when the method of the invention is used to produce ethanol,the plurality of sugar-selective cells includes at least one cell thathas been genetically engineered for enhanced ethanol production.

Production of the biochemical can also optionally be enhanced when atleast one of plurality of sugar-selective cells has been geneticallyengineered to express or overexpress at least one enzyme selected fromthe group consisting of an alcohol dehydrogenase enzyme and a pyruvatedecarboxylase enzyme.

Some embodiments of the method of the invention make use of aninhibitor-selective cell to metabolize one or more inhibitors, such asacetic acid, furfural and hydroxymethyl furfural (HMF), commonly foundin lignocellulosic hydrolysates. The sugar-containing organic materialor biomass, such as the lignocellulosic hydrolysate, is contacted withat least one inhibitor-selective cell under conditions to allow theinhibitor-selective cell to metabolize the inhibitor. Preferably, theinhibitor-selective cell converts the inhibitor into the desiredbiochemical. Contact of the inhibitor-selective cell with thesugar-containing organic material, such as the lignocellulosichydrolysate, can occur prior to contacting the sugar-containing organicmaterial, such as the hydrolysate, with the plurality of sugar-selectivecells, as part of a two stage process, or concurrent with contacting thehydrolysate with the plurality of sugar-selective cells, as part of asingle stage process.

The inhibitor-selective cell is preferably a bacteria or yeast, morepreferably an E. coli cell, wherein at least one gene involved inglucose metabolism has been modified or deleted so as to inhibit orprevent the cell from consuming glucose, and at least one gene involvedin xylose metabolism has been modified or deleted so as to inhibit orprevent the cell from consuming xylose. More preferably, theinhibitor-selective cell has been genetically engineered such that itcannot metabolize any sugar selected from the group consisting ofglucose, xylose, arabinose, galactose and mannose.

More generally, the invention provides a method for producing abiochemical that includes concurrently contacting an organic materialwith a plurality of substrate-selective cells under conditions to allowthe plurality of substrate-selective cells to produce the biochemical.The substrates for which the cells are selective can include any carbonsubstrate, including but not limited to sugars, inhibitors, hydrocarbonsand the like. In an exemplary embodiment, the plurality ofsubstrate-selective cells includes at least one sugar-selective cell andat least one inhibitor-selective cell.

In another aspect, the invention provides a method for removing acetatefrom a mixture, such as organic material, biomass, or a lignocellulosichydrolysate, that includes acetate as well as one or more sugars, suchas glucose and xylose. The mixture is contacted with anacetate-selective cell, preferably an E. coli cell, under conditions toallow the acetate-selective cell to consume acetate. Preferably, atleast one gene involved in glucose metabolism, as well as at least onegene involved in xylose metabolism, have been modified or deleted fromthe acetate-selective cell so as to inhibit or prevent the cell fromconsuming glucose and xylose. More preferably, the acetate-selectivecell has been genetically engineered such that it cannot metabolize anysugar selected from the group consisting of glucose, xylose, arabinose,galactose and mannose. The acetate-selective cell preferably produces abiochemical selected from the group consisting of ethanol, butanol,succinate, lactate, fumarate, pyruvate, butyric acid and acetone.

In another aspect, the invention provides a method for converting asugar-containing organic material, such as a biomass, preferably alignocellulosic hydrolysate, into a biochemical using a two stageprocess. In a first stage, an organic material, such as a biomiass,preferably a lignocellulosic hydrolysate, comprising at least oneinhibitor and a plurality of sugars is contacted with at least onemicroorganism that can only consume an inhibitor, to yield a detoxifiedhydrolysate. In the second stage, the detoxified hydrolysate iscontacted with a plurality of microorganisms, wherein each microorganismindependently consumes only one sugar in the hydrolysate, underconditions to allow the plurality of microorganisms to produce thebiochemical.

It should be understood that the invention encompasses not only themethods described herein, but also, without limitation, thesubstrate-selective cells, and compositions including suchsubstrate-selective cells, as described herein for use in the methods ofthe invention. For example, the invention includes an acetate-selectivecell, preferably an E. coli cell, in which at least one gene involved inglucose metabolism has been modified or deleted so as to inhibit orprevent the cell from consuming glucose, and a modification or deletionof at least one gene involved in xylose metabolism so as to inhibit orprevent the cell from consuming xylose. Preferably the acetate-selectivecell has been genetically engineered such that it cannot metabolize anysugar selected from the group consisting of glucose, xylose, arabinose,galactose and mannose.

Other exemplary compounds include, but are not limited to, a geneticallyengineered microorganism that cannot utilize either glucose or xylose asa carbon source, wherein the corresponding wild-type microorganismutilizes both glucose and xylose as a carbon source. Also included is agenetically engineered microorganism that cannot utilize either glucoseor arabinose as a carbon source, wherein the corresponding wild-typemicroorganism utilizes both glucose and arabinose as a carbon source.Preferably the microorganism is an E. coli, Z. mobilis, C. glutamicum,Lactic Acid Bacteria, S. cerevisiae, P. stipitis or A. monospora. Theinvention further includes a genetically engineered bacterium thatcannot utilize either glucose or xylose as a carbon source. Theinvention further encompasses a genetically engineered bacterium thatcannot utilize either glucose or arabinose as a carbon source. Alsoincluded is a genetically engineered bacterium that cannot utilizeeither glucose or galactose as a carbon source. Preferably the bacteriumis an E. coli, Z. mobilis, C. glutamicum, or Lactic Acid Bacteria.

Yet other exemplary compounds included Pichia stipitis that cannotutilize either glucose or xylose as a carbon source; Pichia stipitisthat cannot utilize either glucose or arabinose as a carbon source;Ambrosiozyma monospora that cannot utilize either glucose or xylose as acarbon source; and Ambrosiozyma monospora that cannot utilize eitherglucose or arabinose as a carbon source.

The invention further includes a composition comprising first and secondsugar-selective cells, wherein the first sugar-selective cellmetabolizes a first sugar that cannot be metabolized by the secondsugar-selective cell, and the second sugar-selective cell metabolizes asecond sugar that cannot be metabolized by the first sugar-selectivecell, and wherein the first and second sugars are independently selectedfrom the group consisting of glucose, xylose, arabinose, galactose, andmannose. Preferably, at least one of the sugar-selective cells canmetabolize only one sugar independently selected from the groupconsisting of glucose, xylose, arabinose, galactose, and mannose.Optionally the composition further includes an inhibitor-selective cell,preferably an inhibitor-selective cell that has been geneticallymodified such that it cannot metabolize any sugar selected from thegroup consisting of glucose, xylose, arabinose, galactose and mannose.Preferred sugar-selective cells include E. coli, Z. mobilis, C.glutamicum, Lactic Acid Bacteria, S. cerevisiae, P. stipitis and A.monospora. Optionally, at least one of the sugar-selective cells hasbeen genetically engineered for enhanced ethanol production. Alsooptionally, at least one of the sugar-selective cells has beengenetically engineered to express or overexpress at least one enzymeselected from the group consisting of an alcohol dehydrogenase enzymeand a pyruvate decarboxylase enzyme. The composition optionally furtherincludes a biomass component such as a lignocellulosic hydrolysate.

Also included in the invention is a composition that includes aplurality of microorganisms of the same species, wherein eachmicroorganism independently utilizes only one sugar, such as glucose,xylose, arabinose, galactose, and mannose, that is present in a biomasssuch as a lignocellulosic hydrolysate. Preferred microorganisms includebacteria and yeast.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims. Unlessotherwise specified, “a,” “an,” “the,” and “at least one” are usedinterchangeably and mean one or more than one.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted concurrently.

It is to be understood that the terms used herein to describe acids (forexample, the term acetate) are not meant to denote any particularionization state of the acid, and are meant to include both protonatedand unprotonated forms of the compound. Thus, the terms acetate andacetic acid refer to the same compound and are used interchangeably.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a two-step process to a) consume inhibitor acetic acid andb) to convert xylose and glucose concurrently into a desired product.

FIG. 2 shows batch aerobic fermentation of Escherichia coli MG1655 on amixture of glucose (∇) and xylose (Δ). The OD () was measured over thecourse of fermentations.

FIG. 3 shows batch aerobic fermentation of single Escherichia colistrains on a mixture of glucose (□) and xylose (Δ). The OD () wasmeasured over the course of fermentations inoculated with A) ZSC113 onlyand B) ALS1008 only.

FIG. 4 shows batch aerobic co-fermentation of Escherichia coli strainsZSC113 and ALS1008 on a mixture of glucose (□) and xylose (Δ). The OD() was measured over the course of the fermentation.

FIG. 5 shows fed-batch aerobic co-fermentation of Escherichia colistrains ZSC113 and ALS1008 using a feed containing a varying mixture ofglucose (dotted lines) and xylose (dashed lines). The OD (), theconcentrations of glucose (□) and xylose (Δ), and the fraction of thetotal cell population which is ZSC113 (▾) were measured over the courseof the fermentation.

FIG. 6 shows batch anaerobic fermentation of Escherichia coli MG1655 ona mixture of glucose (□) and xylose (Δ). After 7 h (h, hours) of aerobicgrowth, additional xylose and glucose were added and anaerobicconditions commenced. The concentrations of formate (▴), lactate (▪),succinate (◯), acetate (♦) and ethanol (⋄) were measured over the courseof the anaerobic phase.

FIG. 7 shows batch anaerobic fermentation of single Escherichia colistrains on a mixture of glucose (□) and xylose (Δ). After 7 h of aerobicgrowth, xylose (for ZSC113) or glucose (for ALS1008) was added andanaerobic conditions commenced. The concentrations of formate (▴),lactate (▪), succinate (◯), acetate (♦) and ethanol (⋄) were measuredover the course of the anaerobic phase previously inoculated with A)ZSC113 only and B) ALS1008 only.

FIG. 8 shows batch anaerobic co-fermentation of Escherichia coli strainsZSC113 and ALS1008 on a mixture of glucose (□) and xylose (Δ). After 7 hof aerobic growth, xylose and glucose were added and anaerobicconditions commenced. The concentrations of formate (▴), lactate (▪),succinate (◯), acetate (♦) and ethanol (⋄) were measured over the courseof the anaerobic phase.

FIG. 9 shows batch aerobic fermentation of Escherichia coli ALS1060(knockouts in ptsG manZ glk xylA) on a mixture of glucose (□), xylose(Δ) and acetate (▴). The OD () was measured over the course of thefermentation time.

FIG. 10 shows batch aerobic-anaerobic process of Escherichia coliALS1074 on a mixture of glucose (▪) and xylose (▴). After 8 h of aerobicgrowth, the culture was switched to anaerobic conditions as indicated.The OD () and lactate concentration (⋄) were measured over the courseof the fermentation.

FIG. 11 shows batch aerobic-anaerobic process of Escherichia coliALS1073 on a mixture of glucose (▪) and xylose (▴). After 8 h of aerobicgrowth, the culture was switched to anaerobic conditions as indicated.The OD () and lactate concentration (⋄) were measured over the courseof the fermentation.

FIG. 12 shows batch aerobic-anaerobic process of two Escherichia colistrains ALS1073 and ALS1074 on a mixture of glucose (▪) and xylose (▴).After 8 h of aerobic growth, the culture was switched to anaerobicconditions as indicated. The OD () and lactate concentration (⋄) weremeasured over the course of the co-fermentation.

FIG. 13 shows batch aerobic-anaerobic process of two Escherichia colistrains ALS1073 and ALS1074 on a mixture of glucose (▪) and xylose (▴).At the start of the process the bioreactor was inoculated with ALS1073,and after two hours the bioreactor was inoculated with ALS1074. After8.5 h of aerobic growth, the culture was switched to anaerobicconditions as indicated. The OD () and lactate concentration (⋄) weremeasured over the course of the co-fermentation.

FIG. 14 shows batch aerobic-anaerobic process of two Escherichia colistrains ALS1073 and ALS1074 on a mixture of glucose (▪) and xylose (▴).At the start of the process the bioreactor was inoculated with ALS1073,and after 3.1 hours the bioreactor was inoculated with ALS1074. After9.1 h of aerobic growth, the culture was switched to anaerobicconditions as indicated. The OD () and lactate concentration (⋄) weremeasured over the course of the co-fermentation.

FIG. 15 shows aerobic batch culture of E. coli MG1655 (hollow symbols)and ALS1060 (solid symbols) on BA10 medium which contains acetate (▴, Δ)as the sole carbon source. The OD (, ◯) was measured over the course ofthe processes.

FIG. 16 shows aerobic batch culture of E. coli ALS1060 on BA10 mediumwith 20 g/L glucose. Glucose (□), acetate (▴) and the OD () weremeasured over the course of the process.

FIG. 17 shows aerobic batch culture of E. coli ALS1060 on BA10 mediumwith 10 g/L xylose. Xylose (⋄), acetate (▴) and the OD () were measuredover the course of the process.

FIG. 18 shows aerobic batch culture of E. coli ALS1060 on BA10 mediumwith 20 g/L glucose and 10 g/L xylose. Glucose (□), xylose (⋄), acetate(▴), and the OD () were measured over the course of the process.

FIG. 19 shows aerobic batch culture of E. coli ALS1122-Kan on BA10medium with 20 g/L glucose and 10 g/L xylose. Glucose (□), xylose (⋄),acetate (▴), and the OD () were measured over the course of theprocess.

FIG. 20 shows growth and substrate consumption of E. coli KD777 on amixture of xylose (▪) and glucose (Δ). The OD (◯) was measured over thecourse of the processes.

FIG. 21 shows growth and substrate consumption of E. coli KD777 on amixture of arabinose (▾) and glucose (Δ). The OD (◯) was measured overthe course of the processes.

FIG. 22 shows growth and substrate consumption of E. coli KD915 on amixture of xylose (▪) and glucose (Δ). The OD (◯) was measured over thecourse of the processes.

FIG. 23 shows growth and substrate consumption of E. coli KD915 on amixture of arabinose (▾) and glucose (Δ). The OD (◯) was measured overthe course of the processes.

FIG. 24 shows growth and substrate consumption of E. coli KD777 on amixture of xylose\ and glucose. Xylose was reintroduced into the mediumafter the initial xylose had been exhausted. OD: ◯; xylose: ▪; glucose:Δ.

FIG. 25 shows growth and substrate consumption of E. coli KD777 on amixture of arabinose and glucose. Arabinose was reintroduced into themedium after the initial arabinose had been exhausted. OD: ◯; arabinose:▾; glucose: Δ.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention provides a novel biological method that permitsefficient conversion of a complex organic material to a desiredbiochemical product. The method of the invention allows multiple sugarsin a complex mixture to be consumed concurrently and independently. Eachsugar is converted into the desired product at relatively high yield,and the process can adapt to fluctuating sugar concentrations withoutleading to the accumulation of any one sugar. Moreover, as describedfurther below, the method can readily be extended to remove inhibitorycompounds from hydrolysates.

The complex organic material that serves as the “input” for the methodof the invention is preferably a biomass, more preferably alignocellulosic biomass. The term “biomass” can include any plant,vegetation, or waste product that can be used as a fuel or used as anenergy source. Cellulosic or lignocellulosic biomass is typically aplant biomass composed of cellulose, hemicellulose and/or lignin, oftenwith smaller amounts of proteins, lipids (fats, waxes and oils) and ash.It should be noted that the terms “cellulosic biomass” and“lignocellulosic biomass” are used interchangeably herein. In somecellulosic biomasses, roughly two-thirds of the dry mass of cellulosicmaterials are present as cellulose and hemicellulose. Lignin makes upthe bulk of the remaining dry mass. Cellulosic biomass feedstocksinclude agricultural plant wastes (e.g., corn stover, leaves and stalks,cereal straws, sugarcane bagasse), plant wastes from industrialprocesses (e.g., wood residues, sawdust, paper pulp, wood chips,municipal paper waste) and energy crops grown specifically for fuelproduction, such as switchgrass. The method of the invention can also bepracticed using crops and feedstocks such as corn, sugarcane, wheat orbarley, and grasses such as switchgrass and hemp. Any organic materialthat contains a plurality of sugars, whether naturally occurring orsynthesized, can serve as the “input” for the method of the invention.Preferably, the organic material, such as the biomass, is hydrolyzed.The principal monomeric sugars found in the lignocellulosic hydrolysateare glucose, xylose, mannose, galactose and arabinose. These sugars havebeen found up to the following percentages in various lignocellulosichydrolysates: arabinose, 17%; galactose, 15%; glucose, 50%; mannose,36%; xylose, 74%.

The organic material is converted into the desired biochemical productor products (the “outputs” of the method) by way of a biologicalprocess. Efficient conversion of inputs to desired outputs isaccomplished by utilizing a plurality of genetically engineered“substrate-selective” cells, each of which has been independentlymetabolically engineered to result in enhanced consumption of oneparticular substrate in relation to another, as described in more detailbelow. Examples of biochemical products include, but are not limited to,ethanol, butanol, succinate, lactate, fumarate, pyruvate, butyric acidand acetone. Other biochemicals that can be produced using thebiological method of the invention include lysine, gluconate, citrate,malate, and whole-cell yeast biomass. Preferably, the method of theinvention produces ethanol.

Importantly, the invention does not rely on a single organism toaccomplish all the process design goals required for biomass, e.g.,lignocellulosic conversion; rather, multiple microbial strains are usedto accomplish tasks efficiently and independently. Note that competitionis minimized or preferably absent in the co-culture contemplated by theinvention. Competition involves multiple species competing for the samesubstrate. In the present invention, the strains each “seek” only theirspecific substrate or substrates. Thus, the various shortcomings whichresult from competition found in a traditional “mixed-culture”bioprocess are reduced or avoided. The present invention can be viewedas an engineered microbial system. Additionally, our approach should notbe confused with “classic consortium” strategies which involve usingdifferent organisms, such as using mixed cultures of P. stipitis and S.cerevisiae, to make ethanol. The problem with these classic consortiumstrategies is that the different microorganisms affect each other'sgrowth, and the system cannot be controlled if one strain still consumesmultiple sugars. Because the strains used in the substrate-selectiveconsortium approach of the invention are substrate-selective andpreferably isogenic, the problems associated with using differentspecies or genera are reduced or avoided.

In one embodiment, the method of the invention encompasses concurrentlycontacting an organic material, preferably a hydrolyzed biomass, with aplurality of substrate-selective cells, under conditions to allowformation of a desired product. The organic material is preferably mixedwith two or more different substrate-selective cells at the same time,in a co-culture, in a single bioreactor. The plurality ofsubstrate-selective cells preferably includes at least one, preferablyat least two, cells that are selective for sugars (sugar-selectivecells). Preferably, the sugar-selective cells include at least onehexose-selective (HSC), at least one pentose-selective cell (PSC), or acombination thereof. In a particularly preferred embodiment, the methodof the invention concurrently utilizes at least one hexose-selectivecell and at least one pentose-selective cell.

Optionally, the method of the invention further includes the use of atleast one substrate-selective cell that is an inhibitor-selective cell(ISC). The inhibitor-selective cell can be contacted with the hydrolyzedbiomass either prior to, or concurrently with, contacting the biomasswith the plurality of sugar-selective cells.

In another embodiment, the invention encompasses contacting a hydrolyzedbiomass with at least one sugar-selective cell and at least oneinhibitor-selective cell, under conditions to allow formation of adesired product. Contacting the hydrolyzed biomass with the differentsubstrate-selective cells can occur concurrently or sequentially. Ifsequential, the hydrolyzed biomass is preferably first contacted withthe inhibitor-selective cell.

A hexose-selective cell used in the method of the invention ispreferably a glucose-selective cell, but can include any otherhexose-selective cell such as cells that are selective for glucose,mannose, galactose, allose and fructose. A pentose-selective cell usedin the method of the invention is preferably a xylose-selective cell,but can include any other pentose-selective cell such as cells that areselective for xylose, arabinose, ribose, and ribulose.

A preferred inhibitor-selective cell for use in the method of theinvention is preferably an acetate-selective cell (ASC). Acetateconsumption in E. coli is mediated by acetyl CoA synthase (acs),isocitrate lyase (aceA), malate synthase (aceB), acetate kinase (ackA)and isocitrate dehydrogenase (icdA). Many other organisms Saccharomycescerevisiae and Pseudomonas spp., Pichia stipitis, Bacillus cereus, haveat least some of these enzymes to degrade acetate however it should benoted that the names of the genes are usually different. For example, inS. cerevisiae the gene encoding for isocitrate lyase is named ICL1. Apreferred acetate-selective bacterial cell has deletions in genesinvolved in the uptake of the all the competing substrates: glucose,mannose, galactose, xylose and arabinose. To that end, the genes manZ,ptsG, glk, xylA, galK, araA are preferably knocked out to yield anacetate-selective cell. Optionally, other genes involved in one or morebacterial phosphotransferase systems (PTS) are knocked out to yield theacetate-selective cell, for example malX from the maltose PTS, fruAandfruB from the fructose specific PTS, the bgl operon (including butnot limited to bglF, bglC, bglS and bglB), and the crr gene. The crr(carbohydrate repression resistance) gene is also known as PTS enzymeIIAGlc. Crr is an important component in glucose uptake through thewell-characterized phosphoenolpyruvate:carbohydrate phosphotransferasesystem, functions a phosphocarrier for glucose transport, and canregulate rpoS expression. A particularly preferred acetate-selectivecell is one containing knockouts of at least manZ, ptsG, glk, xylA, andcrr.

To our surprise, Pseudomonas was found to grow particularly well onacetate and thus is a preferred for use as an acetate-selective cell.

Optionally, an inhibitor selective cell can be engineered to consume twodifferent inhibitors, such as both acetate and furfural. Alternatively,a plurality of inhibitor selective cells can be utilized in the methodof the invention. For example, two different inhibitor selective cells,one that is selective for acetate, and another that is selective forfurfural, can used in the method of the invention. The acetate-selectivestrain would consume acetate and not furfural (i.e., it is engineeredsuch that it lacks one or more furfural degradation genes), and thefurfural-selective strain would consume furfural and not acetate, forexample by knocking out the acetate degradation genes acs and ackA.

As described in more detail below, the inhibitor-selective cell can becontacted with the hydrolyzed biomass either prior to, or concurrentwith at least one, preferably at least two, sugar-selective cells.

It should be understood that the terms “substrate-selective cell,”“sugar-selective cell,” “hexose-selective cell,” “pentose-selectivecell,” “inhibitor-selective cell,” “glucose-selective cell,”“xylose-selective cell,” “acetate-selective cell,” and the like are notintended to refer to just a single, physical cell, but include many suchcells, as in a typical cell culture. Thus, a mixture according to theinvention that contains, for example, one “xylose-selective cell” andone “glucose-selective cell” means a co-culture of xylose-selectivecells and glucose-selective cells. Likewise, a mixture that contains two“hexose-selective cells” means a co-culture of first hexose-selectivecells (e.g., glucose-selective cells) and second (different)hexose-selective cells (e.g., galactose-selective cells).

The invention includes not only a method of producing a desired productfrom a biomass using a plurality of substrate-selective cells, but alsothe various substrate-selective cells, alone or in combination, asdescribed herein. More particularly, the invention includes acomposition that includes a plurality of substrate-selective cells and,optionally, a biomass, such as a lignocellulosic biomass or hydrolysate.

Cells

Cells useful in the method of the invention include animal, plant,yeast, protozoan, and bacterial cells. Bacterial cells and yeast cellsare in common use for large-scale industrial fermentations and arepreferred. Examples of preferred bacterial cells for use in the methodof the invention include Escherichia coli, Zymomonas mobilis,Corynebacterium glutamicum, Lactic Acid Bacteria such as Lactobacillusor Lactococcus, Pseudomonas spp. and Bacillus spp. such as B. subtilisand B. cereus. Lactic Acid Bacteria have several characteristics whichmake them well suited for the biological production of biochemicalproducts. They are natively tolerant of the low pH levels, increasedtemperatures, and ethanol concentrations of hydrolysates, and aretherefore less susceptible than other bacteria to fermentationinhibition. Preferred yeast cells include Saccharomyces cerevisiae,Pichia stipitis and Ambrosiozyma monospora. While cells of single cellmicroorganisms such as bacteria, yeast and protozoa are preferred, themethod can also be practiced using animal or plant cell cultures derivedfrom multicellular organisms.

Substrate Selectivity

Metabolic engineering involves the targeted and purposeful alteration(using genetic engineering techniques) of an organism's metabolicpathways to redesign them to utilize different proportions orcombinations of substrates, or to produce different proportions orcombinations of products. In broad terms, metabolic engineeringencompasses genetically overexpressing particular enzymes at selectedpoints in a metabolic pathway, and/or blocking the synthesis of otherenzymes, to overcome or circumvent metabolic “bottlenecks.” A goal ofmetabolic engineering is to optimize the rate and conversion of asubstrate into a desired product.

The present invention employs a plurality of genetically engineeredmicroorganisms which overcomes the numerous limitations currentlyencountered in the field when utilizing a single microorganism. Cellsuseful in the present invention have been metabolically engineered toresult in enhanced consumption of one particular substrate in relationto another. At least one metabolic pathway of the cells has beendisrupted or altered. In the context of the present invention,disruption or alteration of a metabolic pathway encompasses disruptionin the cellular process for uptake of the substrate from theextracellular environment that reduces or eliminates the ability of thecell to internalize the substrate, as well as a disruption or alterationof an intracellular process involve in the utilization of the substrate.In short, the cells used in the invention are metabolically engineeredto be “substrate-selective.”

A cell is “substrate-selective” when it preferentially utilizes a singleorganic substrate as a carbon source. Carbohydrates (sugars) representthe most common carbon substrates utilized by bacteria and yeast, butmany other organic substrates can be used as carbon sources includinghydrocarbons, lipids, proteins, and small organic molecules such asacids, esters and the like. It should be understood thatsubstrate-selective cells of the invention include cells that have beenengineered to preferentially utilize a particular sugar, inhibitormolecule, hydrocarbon or any other carbon source, without limitation.Such cells are readily identified, designed and selected for use in themethod of the invention by one of skill in the art based upon thecomposition of the organic material to be metabolized, and the nature ofthe desired product.

One way a cell can be made substrate-selective for a particularsubstrate is by modifying it, typically through genetically engineering,to reduce or eliminate the cell's ability to metabolize at least onecompeting substrate. For example, a cell that selectively metabolizesglucose can be made by reducing or eliminating the metabolism of any orall competing substrates such as xylose, galactose, and arabinose. Morespecifically, a cell that normally consumes both glucose and xylose canbe made glucose-selective by reducing or eliminating the cell's abilityto metabolize xylose, and xylose-selective by reducing or eliminatingthe cell's ability to metabolize glucose. Likewise, a cell that normallyconsumes glucose, xylose and arabinose can be made glucose-selective byreducing or eliminating xylose metabolism, arabinose metabolism, or,preferably, both; can be made xylose-selective by reducing oreliminating arabinose metabolism, glucose metabolism, or, preferablyboth; and can be made arabinose-selective by reducing or eliminatingglucose metabolism, xylose metabolism, or, preferably, both. Moregenerally, if a cell can metabolize n sugars (where n>1), the cell canbe made selective for a particular sugar by reducing or eliminating themetabolism of at least 1 other sugar, up to n−1 other sugars in the mostpreferred embodiment. A plurality of sugar-selective cells useful in themethod of the invention thus includes at least a first sugar-selectivecell that metabolizes a first sugar that cannot be metabolized by asecond sugar-selective cell, and a second sugar-selective cell thatmetabolizes a second sugar that cannot be metabolized by the firstsugar-selective cell.

These genetic modifications preferably result in a strain that will onlyutilize a selected sugar (such as glucose, xylose, arabinose, galactose)or inhibitor (such as acetic acid or a furfural), a strain that can beconsidered to have “substrate-selective uptake” since it is selective inwhat compound it is able to consume. For example, by deleting a key genein the xylose uptake pathway, a strain of bacteria is constructed whichis unable to consume xylose, thereby allowing glucose to serve as thespecified substrate. Placed in a fermenter containing xylose andglucose, such a strain would leave the xylose unutilized and becompletely unaffected by the presence of xylose. Similarly, a strain canbe constructed which is unable to consume glucose. Placed in a fermenterwith xylose and glucose, such a strain would be unable to utilize theglucose, and thereby consume only xylose.

Example 16 shows a preferred substrate selective cell that has beenengineered to selectively metabolize xylose and/or arabinose in thepresence of glucose.

Advantageously, a substrate-selective cell functions independently ofother (different) substrate-selective cells, as well as independently ofother substrates present in the mixture. Placed together inco-fermentation in a fermenter containing glucose and xylose, eachstrain would be expected to act optimally on just one of these sugarsand be unaffected by the presence of the other sugar or the otherorganism. For example, a glucose specific microorganism could beengineered by eliminating the pathways necessary for metabolism ofarabinose, galactose and xylose.

Additionally, as discussed in more detail below, the metabolic pathwaysof each strain could be further engineered to optimize yield andproductivity of a particular product, such as ethanol.

A reduction or elimination in the cell's ability to metabolize aparticular substrate can be detected in any convenient manner. Forexample, if a gene has been physically disrupted, e.g., by mutagenesis,deletion, or the like, the disruption can be detected using DNAsequencing or other routine DNA detection procedures. Phenotypically, areduction or elimination in the cell's ability to metabolize aparticular substrate can be detected by comparing the cell's metabolicbehavior relative to a mixture of sugars before and after themodification. If the metabolism of at least one sugar is essentiallyunchanged, but with respect to a second sugar if that sugar ismetabolized slower or not at all, one can conclude that there has been areduction or elimination of the cell's ability to metabolize the secondsugar.

Preferably, the pathway(s) for metabolizing one or more competingsubstrates in the substrate-selective cell is/are completely disrupted(e.g., via a “knockout” of an essential gene), such that the competingsubstrate or substrates otherwise metabolized by the cell are notmetabolized at a detectable level. A knockout of a competing metabolicpathway can be accomplished by modifying or deleting one or more genesinvolved in metabolizing the competing substrate, as necessary, so as toinhibit or prevent the cell from metabolizing the competing substrate.

Methods of disrupting or altering metabolic pathways in bacteria,plants, and animals to reduce or eliminate a cell's ability tometabolize at least one carbon substrate are routine and well known inthe art. Once a molecular target involved in the substrate's metabolismhas been identified, disruption of a metabolic pathway can be effectedat any level of gene expression (e.g., DNA replication, transcription ortranslation), or post-translationally. For example, enzymatic functioncan be inhibited when the enzyme is targeted by a molecular inhibitor,such as an antibody or a small molecule inhibitor. Translation of an RNAmessage into an enzyme can be disrupted, for example, by introducing asmall interfering RNA, a short-hairpin RNA, or a hybridization probeinto the bacteria, plant, or animal cell. Transcription of a geneencoding an enzyme can be disrupted, for example, by targeting the genewith a molecular inhibitor or physically altering the gene to inhibit,prevent or confound gene replication or transcription. Cells can bemetabolically engineered through the introduction of polynucleotides, aswell as the directed mutagenesis of coding regions. Common genedisruption techniques include mutagenesis, gene deletion or knock-out,and heterologous gene transformation. Such methods are well known in theart; see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual.,Cold Spring Harbor Laboratory Press (1989), and Methods for General andMolecular Bacteriology, (eds. Gerhardt et al.) American Society forMicrobiology, chapters 13-14 and 16-18 (1994).

A particularly useful method for metabolically engineering asubstrate-selective cell is to “knock out” an essential gene in themetabolic pathway of a competing substrate. A knock-out technique can beused to render a cell unable to utilize a selected sugar as a carbonsource. A bacterial strain that is unable to use glucose as a carbonsource can be constructed by knocking out the genes encoding forglucosephosphotransferase (ptsG), mannosephosphotransferase (manZ) andglucokinase (glk) (see Example 1). The resulting strain may still beable to metabolize xylose and other sugars, depending on what otherfunctioning metabolic pathways it contains, but not glucose or mannose.

Molecular Targets for Generating Cells that Cannot Utilize One or MoreSugars as a Carbon Source

The molecular targets of metabolic engineering that will yield asubstrate-selective cell for use in the method of the inventionnaturally depend on the nature of the organism, as well as the substratewhose metabolism is to be disrupted.

Particularly preferred targets for metabolically engineering cellsuseful in the method of the present invention are molecules involved incarbohydrate transport and metabolism. A metabolic pathway involved inthe metabolism of a selected sugar, for example, can be disrupted byaffecting the production or activity of one or more enzymes required,either directly or indirectly, to convert the substrate into theproduct. Alternatively, knocking out the production of a receptormolecule needed for uptake or transport of the substrate from theextracellular environment also reduces or eliminates a cell's ability tometabolize the substrate.

Constructing Bacterial Strains that are Incapable of Utilizing SpecificSugars

The phosphotransferase systems of bacteria can translocate a wide rangeof carbon sources into the cytoplasm of bacteria. The overall processcan be represented asphosphoenolpyruvate+carbohydrate→pyruvate+carbohydrate-P where thecarbohydrate is phosphorylated and translocated across the cytoplasmicmembrane so it can be catabolyzed. There are many phosphotransferasesystems which can translocate a wide variety of carbohydrates. Whilemost carbohydrates are translocated by a specific phosphotransferasesystem (PTS), glucose is unusual in that it can be translocated by anyof three different phosphotransferase systems: a glucose-specificglucose PTS that can only translocate glucose, a general glucose PTSthat can translocate ribitol, sorbose and other carbohydrates inaddition to glucose, and the mannose PTS which can translocate glucosein addition to mannose. Thus to inhibit or prevent a bacterium frombeing able to utilize glucose as a carbon source, the glucose-specific,the general glucose, and the mannose phosphotransferase systems must allbe knocked out. Optionally, one or more additional genes involved inother PTSs can be knocked out to further reduce glucose consumption ifdesired, for example malX from the maltose PTS, fruA from the fructosespecific PTS, the bgl operon, and the crr gene.

Some carbohydrates must enter the cell via a periplasmic bindingprotein-dependent (BPD) ABC transporter system where the carbohydrate istranslocated across the cytoplasmic membrane and subsequentlyphosphorylated so it can be catabolyzed. The periplasmic BPD ABCtransporter systems all utilize a periplasmic binding protein, an inneror cytoplasmic membrane protein and an ATPase protein to facilitatetransport.

The sugars that comprise lignocellulosic hydrolysates include arabinose,galactose, glucose, mannose, and xylose. The great majority of bacteriacan utilize these sugars as their primary carbon source. Escherichiacoli as well as many of the Lactic Acid Bacteria can utilize all ofthese sugars. Bacterial strains that are unable to utilize arabinose,galactose, mannose, and xylose as carbon sources can be constructed byeliminating one gene from the respective sugar pathway as indicated inthe following table. The exception is glucose: to construct a bacterialstrain that is incapable of utilizing glucose, either the manX, manY ormanZ gene must be eliminated in addition to the glk and ptsG genes.

Examples of Pathways for Sugar Utilization in Bacteria

Enzyme Gene Reaction catalyzed Pathway Arabinose utilizationArabinose-Binding araF Arabinose transport Arabinose Protein UptakeArabinose Transport araH Arabinose transport Arabinose Membrane ProteinUptake Arabinose ATPase araG Arabinose transport Arabinose ProteinUptake Arabinose araA Arabinose → Ribulose Arabinose IsomeraseCatabolism Ribulokinase araB Ribulose → Ribulose-5-P ArabinoseCatabolism Galactose utilization Galactose Binding mglB Galactosetransport Galactose Protein Uptake Galactose Transport mglC Galactosetransport Galactose Membrane Protein Uptake Galactose ATPase mglAGalactose transport Galactose Protein Uptake Galactokinase galKGalactose → Galactose-1-P Galactose Catabolism Glucose utilizationGlucokinase glk Glucose → Glucose-6-P Glucose Uptake Glucose- ptsGGlucose → Glucose-6-P Glucose phosphotransferase Uptake Enzyme IIMannose PTS manX Glucose → Glucose-6-P Glucose Protein IIA(III) UptakeMannose- manZ Glucose → Glucose-6-P Glucose phosphotransferase UptakeEnzyme IIB Mannose utilization Mannose PTS manX Mannose → Mannose-6-PMannose Protein IIA(III) Uptake Pel Protein manY Mannose → Mannose-6-PMannose Uptake Mannose- manZ Mannose → Mannose-6-P Mannosephosphotransferase Uptake Enzyme IIB Xylose utilization Xylose ProtonxylE Xylose transport Xylose Symport Protein Uptake Xylose IsomerasexylA Xylose → Xylulose Xylose Catabolism Xylulokinase xylB Xylulose →Xylulose-5-P Xylose Catabolism

A cell that cannot utilize glucose as a carbon source can be created,for example, by disrupting the following three genes: ptsG; manZ, manYor manX; and glk. Such a strain will not be able to use mannose, either.

A cell that cannot use xylose as a carbon source can be created, forexample, by disrupting one of the following genes: xylA, xylB, or xylE.

A cell that cannot utilize arabinose as a carbon source can be created,for example, by disrupting one of the following genes: araA, araB, araF,araH, or araG.

A cell that cannot use galactose as a carbon source can be created, forexample, by disrupting one of the following genes: galK, mglB, mglC, ormglA.

A cell that cannot use mannose as a carbon source can be created, forexample, by disrupting one of the following genes: manX, manY or manZ.

Examples of Substrate-Selective Strains Useful in the Method of theInvention Include:

-   -   a) glucose-selective strain: mutations in araA galK xylA manZ        (rendering the cell unable to utilize arabinose, galactose,        xylose or mannose as a carbon source)    -   b) xylose-selective strain: mutations in ptsG manZ glk araA galK        (rendering the cell unable to utilize glucose, mannose,        arabinose or galactose as a carbon source)    -   c) arabinose-selective strain: mutations in ptsG manZ glk xylA        galK (rendering the cell unable to utilize glucose, mannose,        xylose or galactose as a carbon source)    -   d) galactose-selective strain: mutations in ptsG manZ glk araA        xylA (rendering the cell unable to utilize glucose, mannose,        arabinose or xylose as a carbon source)    -   e) mannose-selective strain: mutations in araA galK xylA        (rendering the cell unable to utilize arabinose, galactose, or        xylose as a carbon source; this cell can still utilize glucose        as a carbon source as well as mannose).

Optionally, substrate selective strains, including sugar-selectivestrains and inhibitor-selective strains, also include a mutation in thecrr gene.

Metabolically engineered substrate-selective strains may be furtherengineered for accumulation of the desired biochemical. For example, aglucose-selective E. coli cell engineered to accumulate pyruvate maycontain knockouts of the aceEF pps ldhA poxB pflB arcA atpFH genes,resulting in the accumulation of pyruvate when grown in glucose (Zhu etal., 2008, Appl. Environ. Microbiol. 74:6649). Mutations in the arcA andatpFH genes, however, may be omitted from the analogouspentose-selective strain, since pentoses do not provide the cell with asmuch ATP as glucose, and these two mutations reduce the biomassgeneration from glucose. A sugar-selective E. coli cell engineered toaccumulate succinate, on the other hand, may contain knockouts in thepflB, ldhA and ptsG genes and preferably is engineered to express thepyc (pyruvate carboxylase) gene, which is not endogenous to E. coli (seeVemuri et al., 2002, Appl Environ Microbiol. 68(4):1715-27).

Constructing Yeast Strains that are Incapable of Utilizing SpecificSugars

While many yeasts can utilize all of the sugars found in lignocellulosichydrolysates, the notable exception is Saccharomyces cerevisiae which isunable to use pentose sugars as a carbon source. S. cerevisiae lacksseveral of the key enzymes involved in arabinose and xylose utilization.Yeast strains that are unable to utilize arabinose, galactose, orxylose, can be constructed by eliminating one gene from the respectivesugar pathway as indicated in the following table. To construct a yeaststrain that is incapable of utilizing glucose, either the HXT1, HXT2,HXT3, HXT4, HXT6, HXT7, and SNF3 genes or the GLK1, HXK1, and HXK2 genesmust be eliminated.

Examples of Pathways for Sugar Utilization in Yeast

Enzyme Gene Reaction catalyzed Pathway Arabinose utilization AldoseReductase GRE Arabinose → Arabinitol Arabinose CatabolismArabinitol-4-Dehydrogenase LAD1 Arabinitol → Xylulose ArabinoseCatabolism Xylulose Reductase ALX1 Xylulose → Xylitol ArabinoseCatabolism Xylitol Dehydrogenase XYL2 Xylitol → Xylulose XyloseCatabolism Xylulokinase XKS1 Xylulose → Xylulose-5-P Xylose CatabolismGalactose utilization Galactose Permease GAL2 Galactose transportGalactose Uptake Galactokinase GAL1 Galactose → Galactose-1-P GalactoseCatabolism Galactose-1-P Uridyl Transferase GAL7 Galactose-1-P →Glucose-1-P Galactose Catabolism UDP-Glucose-4-Epimerase GAL10UDP-Glucose → UDP-Galactose Galactose Catabolism Glucose utilizationLow-affinity glucose transporter HXT1 Glucose transport Glucose UptakeHigh-affinity glucose transporter HXT2 Glucose transport Glucose UptakeLow-affinity glucose transporter HXT3 Glucose transport Glucose UptakeHigh-affinity glucose transporter HXT4 Glucose transport Glucose UptakeHigh-affinity glucose transporter HXT6 Glucose transport Glucose UptakeHigh-affinity glucose transporter HXT7 Glucose transport Glucose UptakePlasma membrane glucose sensor SNF3 Glucose transport Glucose UptakeGlucokinase GLK1 Glucose → Glucose-6-P Glucose Uptake Hexokinaseisoenzyme 1 HXK1 Glucose → Glucose-6-P Glucose Uptake Hexokinaseisoenzyme 2 HXK2 Glucose → Glucose-6-P Glucose Uptake Xylose utilizationXylose Reductase XYLI Xylose → Xylitol Xylose Catabolism XylitolDehydrogenase XYL2 Xylitol → Xylulose Xylose Catabolism XylulokinaseXKS1 Xylulose → Xylulose-5-P Xylose Catabolism

A yeast cell that cannot utilize glucose as a carbon source can becreated, for example, by disrupting the following three genes: GLK1,HXK1, and HXK2. A yeast cell that cannot utilize glucose as a carbonsource can also be created by disrupting the following seven genes:HXT1, HXT2, HXT3, HXT4, HXT6, HXT7, and SNF3.

A yeast cell that cannot utilize arabinose as a carbon source can becreated, for example, by disrupting one of the following genes: GRE,LAD1, or ALX1. It is preferable not to delete the XYL2 or XKS1 genessince these genes are also utilized in xylose catabolism.

A yeast cell that cannot utilize galactose as a carbon source can becreated, for example, by disrupting one of the following genes: GAL2,GAL1, GAL7, or GAL10.

A yeast cell that cannot utilize xylose as a carbon source can becreated, for example, by disrupting one of the following genes: XYL1,XYL2, or XKS1.

Optimization of Ethanol Production

The substrate-selective cell used in the method of the inventionincludes at least one metabolic pathway for the production of thedesired product, for example ethanol. If the native, wild-type cell doesnot include an ethanol production pathway, genetic engineering can beused to introduce such a pathway so as to render the cell ethanologenic.If the native cell includes an ethanol pathway, the cell can optionallybe further metabolically engineered to optimize the ethanol productionpathway.

Optionally, the substrate-selective cell of the invention is engineeredto overexpress an alcohol dehydrogenase enzyme and/or a pyruvatedecarboxylase enzyme. Pyruvate is converted by pyruvate decarboxylase toacetaldehyde, which is subsequently converted to ethanol by alcoholdehydrogenase. In some endogenous bacterial systems, the conversion ofpyruvate to ethanol is rate-limiting in the production of ethanol, andresults in excess substrate being converted to other byproducts such asacetic acid and succinic acid. The presence of high activities ofalcohol dehydrogenase enzyme and pyruvate decarboxylase enzymeeffectively increase the organism's ability to produce ethanol in orderto maintain high conversion efficiency and minimize the amount ofbyproduct. One option for introducing these two enzymes into asubstrate-selective cell is to transform the cell with a plasmid, suchas pLOI308 (Ingram et al., 1987, Appl. Environ. Microbiol. 53:2420;Ingram and Conway, 1988, Appl. Environ. Microbiol. 54:397), thatelevates ethanol formation in E. coli. This plasmid uses the Zymomonasmobilis pyruvate decarboxylase and alcohol dehydrogenase B genes.Preferably, the cell is transformed with a “production of ethanol” (PET)operon in which the alcohol dehydrogenase gene and the pyruvatedecarboxylase gene from Z. mobilis have been placed under control of theLac promoter. When expressed together, alcohol dehydrogenase gene andpyruvate decarboxylase cause ethanol to be the major product of E. colifermentations. Alternatively or additionally, the cell can betransformed with a plasmid, such as pTrc99A-pdc.adhB, which containspyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) from Z.mobilis.

Alternatively or additionally, ethanol production can be optimized byknocking out by-product pathways in order to guide additionalsubstrates, and force more carbon, into the ethanol production pathway.By-product pathways genes that can be knocked-out in order to enhanceethanol production include, but are not limited to, pyruvate formatelyase, fumarate reductase, and lactate dehydrogenase.

Biological Production of Ethanol and Other Biochemicals

Biological conversion of organic material, such as a biomass, to adesired product, such as ethanol, can involve one or more of thefollowing steps, in any order: harvesting the biomass, performingseparation or extraction steps on the biomass, pretreating the biomass,hydrolyzing the biomass, contacting the biomass with substrate-selectivecells of the invention under conditions to convert a component of thehydrolysate into a desired product (i.e., fermentation), and subsequentisolation and/or purification of the product.

A biological approach to the production of ethanol typically involvesfour or five stages. A “pretreatment” phase can be used to make thebiomass, including lignocellulosic material such as wood or straw,amenable to hydrolysis. Pretreatment assists in liberating the cellulosefrom the lignin and makes it more accessible to hydrolysis.Pretreatments are done through physical (mechanical) and/or chemicalmeans. Pretreatment techniques include acid hydrolysis, steam explosion,ammonia fiber expansion, alkaline wet oxidation and ozone pretreatment.

Cellulose and hemicellulose molecules are long chains of sugar moieties.Hydrolysis is used to break down the long molecules in the cellulosicbiomass into simple sugars, preferably monomeric sugars, prior tosubjecting the mixture to biological conversion of the sugars into thedesired products. Hydrolysis can be effected using chemical, physical orenzymatic means. The most commonly used method of chemical hydrolysis isacid hydrolysis. Dilute acid can be used under high heat and highpressure, or more concentrated acid can be used at lower temperaturesand pressures. The products from this hydrolysis are neutralized, andmicrobial fermentation according to the present invention can then beused to produce the desired product, such as ethanol. It should be notedthat acid hydrolysis produces several potent inhibitors including aceticacid, furfural and hydroxymethyl furfural (HMF). Advantageously, themixture can be cleared of inhibitors by contacting it with a cell thatis selective for the inhibitor, as a carbon substrate, in accordancewith the method of the present invention.

Alternatively or additionally, an enzymatic hydrolysis process can beused. Cellulose chains can be broken into glucose molecules by cellulaseenzymes. Biomass, including lignocellulosic biomass can be enzymaticallyhydrolyzed at a relatively mild condition (50 deg C. and pH5), thusenabling effective cellulose breakdown without the formation ofbyproducts that would otherwise inhibit enzyme activity.

Optionally, prior to biological fermentation of the mixture inaccordance with the invention, the sugars in the hydrolysate areseparated from residual materials, such as lignin. At this point themixture is subjected to biological production of the product(s) using aplurality of substrate-selective cells in accordance with the invention.The resulting product, such as ethanol or lactate, may then be isolatedand purified. In methods where ethanol is produced, isolation andpurification can be accomplished by distillation.

Biological fermentation, which is sometimes termed “microbialfermentation” when microorganisms are used, yields the desired product,such as ethanol or lactate. It should be noted that the word“fermentation” as used herein is not limited to a strict biochemicalmeaning of an anaerobic process of converting carbon substrates intoacids and alcohols; rather the word “fermentation” is used more broadlyas in the art of industrial microbiology, such that it is meant toencompass any large-scale production of biochemicals usingmicroorganisms. As such, a “fermentation” can be either aerobic oranaerobic. Contacting an organic, sugar-containing material, such as abiomass, preferably a lignocellulosic hydrolysate, with the plurality ofsubstrate-selective cells of the invention under conditions to form aselected product is considered herein to be a “fermentation” regardlessof whether the process is performed under aerobic or anaerobicconditions.

Biological fermentation, during which substrate-selective cells of theinvention are contacted with the biomass hydrolysate under conditions toconvert a component of the hydrolysate into a desired product, may takeany form, without limitation. For example, it may be a batchfermentation, a fed-batch fermentation, a steady-state fermentation or acontinuous fermentation. Fermentation may be done under either aerobicor anaerobic culture conditions, or both in a sequential manner.

Advantageously, use of a plurality of substrate-selective cells allowsbiological fermentations to be customized in response to the particularcomposition of the “inputs” or feed. Different feeds, such as crops,wheatgrass, lignocellulosics and other organic material, contain varyingcompositions of sugars. Volumetric consumption rates for each cell typecan be measured and used to effect an “alignment” of consumption ratesthat permits the most efficient consumption of the available substrates.Optimized utilization of substrates (including sugars and inhibitorcompounds) can be achieved by manipulating the timing of the inoculationof one or more of the substrate-selective cells and/or by manipulatingthe amount or activity (e.g., by upregulating or overexpressing aprotein involved in the consumption of the selected substrate) of theinoculum. Use of a fed-batch fermentation system also allows consumptionrates for each of the substrate-selective cells to be optimized inresponse to a particular feed.

In embodiments of the method which do not employ inhibitor-selectivecells, biological processing of the hydrolyzed biomass is preferablycarried out in a single bioreactor, in a co-fermentation that includesthe plurality of sugar-selective cells and the hydrolyzed biomass. Theplurality of sugar-selective cells operate independently andconcurrently on the hydrolyzed biomass and, as noted elsewhere herein,can be responsive to changes in the relative proportions of the sugarsin the fermenter. In embodiments that employ one or moreinhibitor-selective cells in addition to sugar-selective cells, either asingle stage process (all cells concurrently and independently operatingon the biomass in a single bioreactor) or a dual stage process(inhibitor-selective cells operating on the biomass first, followed bycontact with the sugar-selective cells) can be used, as set forth inmore detail below.

The substrate-selective cells used in a particular stage of thefermentation process according to the invention are preferably eitherall bacterial cells, or all yeast cells. For example, in a dual stageprocess as described in more detail below, acetate-selective E. colicells could be used in Stage 1 in a first reactor, followed by a secondreactor in Stage 2 containing a collection of yeasts having varioussugar-selectivities.

The efficacy of fermentation reactions is further complicated becausehydrolysis of organic material continually generates compounds whichinhibit the subsequent conversion of organic substrates such as sugarsby fermentation. As discussed above, xylose is acetylated inlignocellulose (Timmel, 1967, Wood Sci. Technol. 1:45; Chesson et al.,1993, J. Sci. Food Agric. 34:1330), and therefore acetic acid, aparticularly potent inhibitor of fermentation, is an unavoidable productof lignocellulose hydrolysis. Production of some inhibitors can bereduced by alterations in the hydrolysis process or in the organicmaterial itself (e.g., through genetic engineering). However, it has todate not been feasible to eliminate all inhibitors. Not only must anefficient fermentation system be able to handle a mixture of substrates,but it must also be able to proceed in the fluctuating presence of someinhibitors such as acetic acid without loss of either yield orproductivity. Previous approaches to convert mixtures of sugars andinhibitors have not been able to achieve the rate of conversionnecessary to make the process economically viable.

Thus, in embodiments of the method of invention that utilize aninhibitor-selective cell (ISC), preferably an acetate-selective cell(ASC), either a single stage or a dual stage (two step) fermentation canbe employed.

In a single stage fermentation, the inhibitor-selective cell (e.g., anacetate-selective cell) and at least one, preferably at least two,sugar-selective cells are contacted with the hydrolysate concurrently.

In a dual stage fermentation (FIG. 1), the inhibitor-selective cell isfirst contacted with the hydrolysate in order to metabolize the aceticacid generated during hydrolysis (Stage 1). The hydrolysate is thencontacted with the sugar-selective cells to metabolize the sugarmonomers present in the mixture (Stage 2). Advantageously, theinhibitor-selective cells may lyse after the acetate is fully consumed,thereby providing additional nutrients for the sugar-selective cells.

In a process using acetate-selective cells in Stage 1, it isadvantageous to maintain Stage 1 as a continuous (or fed-batch) processto maintain a zero acetate concentration as a result of substratelimitation. The process of this continuous process is then matched withStage 2, which is preferably conducted batch fermentation, linearfed-batch fermentation, or exponential fed-batch fermentation (Example8). In a preferred embodiment, the two stage process is operates infed-batch or continuous mode under carbon (i.e., acetate) limitation.

Acetate consumption in Stage 1 is preferably performed under aerobicconditions. Stage 2 can be anaerobic or aerobic, depending on the cellsused and the identity of the biochemical being produced. Optimalproduction of the fermentation product ethanol or lactate, for example,during Stage 2 is preferably accomplished under anaerobic conditions.Thus, when the method of the invention is used to produce ethanol orlactate, a dual stage fermentation may be preferable when an optionalinhibitor-selective cell is used.

Optionally, the cultures can be supplemented with additional nutrientsin Stage 1, Stage 2, or both; or in the single stage when a single stageprocess is used.

The utilization of a two stage process that first eliminates thepresence of inhibitors, then converts metabolic substrates into thedesired fermentation product, increases both the rate of conversion andproduct yield while leaving little residual biomass hydrolysate unused.

Thus, as summarized above, and as illustrated and described in moredetail in the following Examples, the present invention provides amethod of concurrent conversion of hexose and pentose sugars which arepresent in biomass such as lignocellulosic hydrolysates obtained fromorganic material. The problem is that a single microorganism is unableto metabolize multiple sugars concurrently. Instead, any givenmicroorganism has a complex regulatory network which forces sugars to bemetabolized sequentially. This sequential nature invariably reduces theoverall rate of a fermentation process to generate the desired product.

Moreover, the fermentation of hexose and pentose sugars is furtherreduced by inhibitory compounds, such as acetate, which are unavoidablein the hydrolysate. Although the inability of microorganisms to utilizemixed sugars in the presence of acetate is most commonly associated withfuel ethanol production, the formation of other fermentation products(butanol, succinic acid, lactate, pyruvate, fumarate etc.) from biomasshydrolysates would similarly be greatly enhanced by a process that couldeliminate acetate and effectively use a multitude of sugars.

Another significant disadvantage in current one-organism processes isthat the metabolic pathways to convert glucose into a desired product atoptimal yield and productivity do not generally correspond to themetabolic pathways to convert xylose into the same product. Ideally, aprocess converting xylose and glucose concurrently into a single productwould permit these pathways to operate independently of one another,with glucose metabolism not influencing xylose metabolism and viceversa.

The invention thus addresses many of the limitations that have plaguedprevious attempts to convert lignocellulosic biomass into ethanol andother desired products. The method is able to simultaneously metabolizea variety of organic substrates, thereby eliminating the growth patterndemonstrated by a single microorganism in which it linearly consumes onesubstrate at a time. Additionally, while employing a number of metabolicalterations that, if made to a single organism, would result in anunstable cell that is prone to spontaneous mutations, these alterationsare made in a plurality of different organisms, yielding a highly stablesystem. Furthermore, each cell within the plurality, by independentlymetabolizing a specific substrate, is able to fully utilize thatsubstrate resulting in maximum yield of a single metabolic product,whereas a single cell attempting to metabolize a number of differentsubstrates often yields a number of different products. By concurrentlymetabolizing multiple organic substrates, inhibitors are depleted,production efficiency is improved, and yields of fermentation productsare enhanced. The process of the present invention is able to handlevarying mixtures of sugars and is also able to contend with thefluctuating presence of some inhibitors such as acetic acid without lossof yield or productivity.

The present invention provides a plurality of cells capable of botheliminating inhibitors and concurrently converting hexose and pentosesugars into a desired fermentation product. The invention is to bebroadly understood as including methods of making and using the variousembodiments of the metabolically engineered cells of the inventiondescribed herein.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Co-Fermentation Strategy to Consume Sugar MixturesEffectively Introduction

We report a new approach for the simultaneous conversion of xylose andglucose sugar mixtures into products by fermentation (Eiteman et al.,2008, J. Biol. Engineering, 2:3). The process simultaneously uses twosubstrate-selective strains, one which is unable to consume glucose andone which is unable to consume xylose. The xylose-selective (glucosedeficient) strain CGSC5457 (ZSC113) has mutations in the glk, ptsG andmanZ genes while the glucose-selective (xylose deficient) strain ALS1008has a mutation in the xylA gene. By combining these two strains in asingle process, xylose and glucose are consumed more quickly than by asingle-organism approach. Moreover, we demonstrate that the process isable to adapt to changing concentrations of these two sugars, andtherefore holds promise for the conversion of variable sugar feedstreams, such as biomass sources including lignocellulosic hydrolysates.

The goal of this study was to characterize xylose-selective andglucose-selective strains of Escherichia coli. Specifically, we set outto construct E. coli strains which independently will only consumexylose or glucose without any loss in consumption rate and which can beused together, acting in concert to consume sugar mixtures effectively.

Materials and Methods Strains

Escherichia coli strains MG1655 (wild-type, F-, λ-) CGSC5457 (ZSC113,lacZ827(UGA) or lacZ82(Am)ptsG22 manZ12 glk-7 relA1 rpsL223(strR)rha-4), DY330 (ΔlacU169 gal490 λc1857 Δ(cro-bioA)), and ALS1008 (MG1655xylA::Tet) were used in this study. CGSC5457 (ZSC113) was obtained fromthe E. coli Genetic Stock Culture (Yale University). DY330 contains alambda cI857 lysogen and is used to induce the genes which constitutethe lambda Red recombination system (Yu et al., 2000, Proc Natl Acad SciUSA. 97:5978).

Generating the xylA::Tet Knockout

The xylA gene which encodes D-xylose isomerase was knocked out using thelambda Red recombination system. Primers were designed which couldamplify the tetA gene and promoter from pWM41 (Metcalf et al., 1996,Plasmid. 35:1.) bracketed by the first and last 50 bases of the xyIAcoding sequence. The tetA gene codes for the tetracycline resistanceprotein. The forward primer 5′ATGCAAGCCTATTTTGACCAGCTCGATCGCGTTCGTTATGAAGGCTCAA AACATCTCAATGGCTAAGGCG3′ (SEQ ID NO:1) contains the first 50 bases of the xylA coding sequencefollowed by bases 1349-1368 of TRN10TETR (Accession Number J01830) frompWM41 while the reverse primer5′TTATTTGTCGAACAGATAATGGTTTACCAGATTTTCCAGTTGTTCCTGGCGGCTGGTTTATGCATATCGC (SEQ ID NO:2) 3′ contains the last 50 bases of thexylA coding sequence followed by bases 3020-3039 of TRN10TETR frompWM41. The bases from pWM41 are underlined in the primers. The twoprimers were used to amplify a 1,791 bp fragment from pWM41 DNA usingthe polymerase chain reaction (PCR) with Pfu polymerase. The resultingDNA was gel-isolated and electroporated into DY330 electrocompetentcells which were prepared as described (Yu et al., 2000, Proc. Natl.Acad. Sci. USA 97:5978). Tet(R) colonies were then selected. Thepresence of the xylA::Tet knockout was confirmed by the inability ofDY330 xylA::Tet to grow in minimal xylose media. ALS1008 (MG1655xylA::Tet) was constructed by transducing xylA::Tet from DY330 xylA::Tetinto MG1655 using P1 transduction.

Growth Conditions

For each bioreactor experiment, a single strain was first grown in atube containing 10 mL BXG medium, then 5 mL transferred to 50 mL BXGmedium in a 250 mL shake flask. All flasks were incubated at 37° C. and250 rpm (19 mm pitch). For those fermentations in which a single strainwas used, when the OD of the shake flask culture reached approximately4, the contents of the shake flask were diluted with BXG medium so that100 mL having an effective OD of 2.0 was used to inoculate thefermenter. For those experiments in which two strains were used in asingle fermentation, the contents of two shake flasks were diluted withBXG medium to 100 mL so that each strain had an effective OD of 2.0(i.e., in the 100 mL volume). Basal medium contained (per L): 13.3 gKH₂PO₄, 4.0 g (NH₄)₂HPO₄, 1.2 g MgSO₄.7H₂O, 13.0 mg Zn(CH₃COO)₂.2H₂O,1.5 mg CuCl₂.2H₂O, 15.0 mg MnCl₂.4H₂O, 2.5 mg CoCl₂.6H₂O, 3.0 mg H₃BO₃,2.5 mg Na₂MoO₄.2H₂O, 100 mg Fe(III)citrate, 8.4 mg Na₂EDTA.2H₂O, 1.7 gcitric acid, and 0.0045 g thiamine.HCl. BXG medium comprised basalmedium with 15 g/L glucose and 8 g/L xylose. Shake flask media wereadjusted to a pH or 7.0 with 20% NaOH.

Fermentation

Batch experiments were carried out in a 2.5 L bioreactor (Bioflow 2000,New Brunswick Scientific Co. Edison, N.J., USA) containing 1.0 L BXGmedium. Throughout aerobic fermentations, air was sparged into thefermenter at a flowrate of 1.0 L/min, and the agitation was 1000 rpm toensure no oxygen limitation. In some experiments an anaerobic phase wasinitiated after aerobic growth. For these cases additional xylose and/orglucose was supplied as reported. To maintain anaerobic conditions,carbon dioxide was provided at a flowrate of 0.2 L/min, and theagitation was 150 rpm.

Fed-batch experiments were carried out in the same vessel initiallycontaining 1.0 L basal medium (no xylose and glucose). Immediately afterinoculation, a feed containing a mixture of xylose and glucose withoutadditional medium components commenced as reported. This medium was fedat an exponentially increasing rate designed to achieve a growth rate of0.1 h⁻¹ for a substrate concentration of 30 g/L.

For all fermentations, the pH was controlled at 6.7 using 15% (w/v)NH₄OH, and the temperature was controlled at 37° C.

Analyses

The optical density at 600 nm (OD) (UV-650 spectrophotometer, BeckmanInstruments, San Jose, Calif.) was used to monitor cell growth, and thisvalue was correlated to dry cell mass. Previously described liquidchromatography methods were used to quantify xylose and glucose (Eitemanet al., 1997, Anal. Chem. Acta 338:69) and other organic compounds(Chesson et al., 1993, J. Sci. Food Agric. 34:1330).

The fraction of the microbial population that constituted each strainwas determined by plating serial dilutions of cultures onto both LB andLB-tetracycline plates.

Results Aerobic Utilization of Xylose/Glucose Mixtures

Escherichia coli ZSC113 and E. coli ALS1008 are unable to consumeglucose and xylose, respectively. The xylose-selective strain ZSC113 hasmutations in the three genes involved in glucose uptake (Curtis andEpstein, 1975 J Bacteriol. 122:1189), rendering it unable to consumeglucose: ptsG codes for the Enzyme IICB^(Glc) of the phosphotransferasesystem (PTS) for carbohydrate transport (Postma et al., 1993 MicrobiolRev. 57:543), manZ codes for the IID^(Man) domain of the mannose PTSpermease (Huber and Erni, 1996, Eur J Biochem. 239:810), glk codes forglucokinase (Curtis and Epstein, 1975 J Bacteria 122:1189). Weconstructed strain ALS1008 which has a knockout in the xylA geneencoding for xylose isomerase, rendering ALS1008 unable to consumexylose. In a medium composed of a mixture of these two sugars, ZSC113would be expected to consume the xylose selectively while ALS1008 shouldexclusively consume the glucose. We first sought to verify theseexpectations in three aerobic batch experiments.

In a first (control) experiment, a defined medium containing both 8 g/Lxylose and 15 g/L glucose was inoculated with a single wild-type strain,MG1655, and grown aerobically (FIG. 2). The glucose/xylose mixture waschosen to reflect the concentrations of glucose and xylose that arefound in typical lignocellulosic hydrolysates. As expected, we observeddiauxic growth as reported by many other studies when a single strain isinoculated into a medium containing two or more carbon sources. Theimportant observations were that glucose and xylose were consumedsequentially, and that the complete consumption of this mixture requiredabout 8.5 hours.

In a second set of aerobic experiments, the same defined mediumcontaining two carbon sources was inoculated with one or the other ofthe two strains, ZSC113 or ALS1008. In the fermenter inoculated withonly ZSC113 (FIG. 3 a), 8 g/L xylose was completely consumed in 7 h andthe OD reached 10, In this case, the concentration of glucose remainedunchanged. In the fermenter containing only ALS1008 (FIG. 3 b), weobserved the complete consumption of 15 g/L glucose in 7.5 h with the ODreaching 15, while the concentration of xylose remained unchanged. Asexpected the two strains each consumed only one of the sugars, leavingthe other carbohydrate unconsumed.

In a third aerobic batch experiment, we inoculated both ZSC113 andALS1008 into a single fermenter containing 8 g/L xylose and 15 g/Lglucose. For this co-culture fermentation, glucose was consumed in 7.5h, and xylose was simultaneously consumed in 7.0 h (FIG. 4). Moreover,the final OD of this mixed culture was about 25, identical to the sum ofthe ODs achieved in the fermentations in which one or the othercarbohydrate was used. Thus, each strain appears to grow and consume itssubstrate independently. The combined process (i.e., consuming bothsugars simultaneously) occurred at the same rate as the two individualprocesses so that each consumption rate was unaffected by the presenceof the other carbohydrate. Compared to the wild-type (single organism)process, this process required about 15% less time to consume the samecarbohydrate mixture aerobically, and moreover each substrate wasconsumed independently. The single-organism process (FIG. 2) wascompletely different than the dual-organism process (FIG. 4) in whichboth carbon sources were consumed simultaneously. No products wereobserved in these batch fermentations.

Aerobic Fed-Batch Utilization of Xylose/Glucose Mixtures

When a microorganism grows in a substrate-limited fashion (for example,in a fed-batch process), the growth rate is controlled by the rate thatthe limiting substrate is supplied. Moreover, the concentration of thatsubstrate remains at zero. In a bioprocess with two substrate-selectiveorganisms which are both under carbon-limiting conditions, each organismshould independently be controlled by and adapt to the quantity of thecarbon source present that it can consume. We wished to test thishypothesis using a fed-batch process in which the two-carbohydrate feedincreased exponentially at a nominal rate of 0.1 h⁻¹, far below themaximum growth rate of either strain. Moreover, in addition to theflowrate exponentially increasing to maintain a fixed specific growthrate, the composition of the feed changed in discrete shifts in order tosimulate a variable concentration that might be encountered in a realprocess. Specifically, for the first 20 h we maintained feedconcentrations at 20 g xylose/L and 30 g glucose/L (20:30). At 20 h,this feed was replaced by feed concentrations of 30:30, at 30 h to30:60, and then finally to 20:60 at 40 h. At 20 h, 30 h, 40 h and 50 hwe determined the fraction of the population which was theglucose-consuming strain ALS1008 (and thus by difference the fractionwhich was the xylose-consuming strain ZSC113).

During the entire fed-batch process, the xylose and glucoseconcentrations in the fermenter remained at zero (FIG. 5), demonstratingthat each substrate individually limited the process. Moreover, thedistribution of the microbial population responded in unison with theshift in substrate concentrations. At 20 h, after the process hadacclimated to a 20:30 xylose:glucose composition (g/L), the populationwas 35% ZSC113 (i.e., the xylose-consuming strain). Ten hours after thefeed composition shifted to 30:30, the population was 50% ZSC113.Similarly, ten hours after the feed composition shifted to 30:60, thepopulation returned to 42% ZSC113, and then ten hours after the feed hadbecome 20:60, the population decreased to 32% ZSC113. These resultsdemonstrate that the process adjusts the distribution of strains tomatch the distribution of substrates.

Anaerobic Product Formation with Xylose/Glucose Mixtures

Wild-type E. coli is a mixed acid fermenter, and generates acetate,lactate, formate, ethanol and succinate under anaerobic conditions, withthe yield of each depending on the strain and carbon source (Clark,1989, FEMS Microbiol. Rev. 63:223). The strains used for this study ofsubstrate-selective uptake did not generate an elevated concentration ofany product during the aerobic studies (beyond the expectation forwild-type strains). ZSC113 and ALS1008 do not have any additionalmutations which would cause the product distribution to be differentfrom the wild-type parent MG1655. We thus wanted to examine how ZSC113and ALS1008 behaved under anaerobic conditions in which products wouldaccumulate. Three experiments were conducted under anaerobic conditions,analogous to those conducted under aerobic conditions previously.

In a first experiment, wild-type MG1655 was grown under aerobicconditions as before to consume 15 g/L glucose and 8 g/L xylose. Whenboth substrates were nearly consumed (after about 8.5 h as shown in FIG.2), we added enough glucose and xylose into the fermenter approximatelyto return both concentrations to their initial levels. Anaerobicconditions were initiated under an atmosphere of 100% CO₂, and theproducts were measured during the anaerobic growth phase (FIG. 6). Inthis culture of a single strain (having an OD of 21) 10 g/L of glucosewas consumed in about 2.5 h (4 g/Lh), equivalent to a specific glucoseconsumption rate of 630 mg/gh. Initially xylose was consumed at a rateof 310 mg/gh (2 g/Lh). However, after 4 h of anaerobic conditions, thexylose consumption rate decreased to less than 150 mg/gh (1 g/Lh), andcontinued to slow. Nearly 4 g xylose/L remained after 9 h of anaerobicconditions. It must be noted that the organism consumed glucose thenxylose in the aerobic phase preceding these anaerobic conditions (asshown in FIG. 2), and therefore at the time of the switch to anaerobicconditions the xylose-consuming pathways were fully induced. Oneexplanation for the substantial decrease in xylose consumption rate isthe sensitivity of xylose-degradation to the presence of acetate aspreviously reported for yeast (Helle et al., 2003, Enzyme MicrobialTechnol. 33(6):786; van Zyl et al., 1991, Enzyme Micro. Technol. 13:82).This explanation is supported by the observation that xylose consumptioncontinued to slow even after glucose was depleted under anaerobicconditions.

In a second experiment, the two substrate-selective strains ALS1008 andZSC113 were grown individually on the mixed substrate medium, the onedepleted substrate added back, and then anaerobic conditions commenced.For the case of the xylose-consuming strain ZSC113, xylose was consumedat a constant rate of 1.4 g/Lh during the anaerobic phase and glucosewas not consumed (FIG. 7 a). This single organism was present only at anOD of 9.5, so that on a specific basis the xylose consumption rate was500 mg/gh, greater than the highest rate observed in the xylose portionof the fermentation using the wild-type MG1655. For the experiment inwhich the glucose-consuming strain ALS1008 was inoculated into the mixedsubstrate medium, glucose was exclusively consumed during the anaerobicphase at a constant rate of 3 g/Lh, and xylose was not consumed (FIG. 7b). In this case, the specific glucose consumption rate was about 770mg/gh, greater than the rate we observed for the wild-type MG1655 duringthe anaerobic phase (i.e., FIG. 6). These two separate fermentationsdemonstrate that the strains will each consume only one substrate underanaerobic conditions, and that they will consume this substrate slightlyfaster on a specific basis than the wild-type strain would underidentical conditions.

In a third experiment we first simultaneously grew both strains in themixed substrate medium under aerobic conditions. At the end of the 7.0 haerobic growth phase, we added enough of both carbohydrates to returnthem to their initial concentrations, and anaerobic conditions wereinitiated. In this process, both xylose and glucose were quicklyconsumed (FIG. 8). Although we did not measure the proportion of the twostrains, from previous aerobic results using one substrate (i.e., FIG.3), we estimate that the OD of ZSC113 was about 13 and the OD of ALS1008was about 9. Over the first two hours of the anaerobic phase, the xyloseconsumption rate was therefore about 475 mg/gh, while the glucoseconsumption rate was about 1300 mg/gh. The key point in these results isthat the two-strain process is much faster than an otherwise identicalsingle-strain process.

The products formed from the two-sugar fermentation were the same asthose generated during either one of the single-sugar fermentations,although the distribution of products changed slightly. For example, thesuccinate yield from xylose using ZSC113 was 0.48 mol/mol, while thesuccinate yield from glucose using ALS1008 was 0.30 mol/mol. From thesugar mixture, the observed succinate yield by two organisms was 0.41mol/mol sugar consumed, a value between the yields of the individualstrains on the two substrates. For all three cases, formate wasgenerated with the highest yield (1.24 mol/mol glucose, 1.58 mol/molxylose, and 1.46 mol/mol sugar mixture), and lactate was generated theleast.

One limitation of this study was that high concentrations of acidproducts are known to inhibit growth and substrate consumption rates.This phenomenon would tend to affect the mixed culture more than eithersingle-sugar culture, since for the former case both sugars wouldquickly be converted into more mixed acid products. Thissubstrate-selective approach may perform significantly better forstrains specifically designed to accumulate a single product such asethanol which does not cause acid inhibition.

Discussion

The process described in this study offers a new approach for thesimultaneous conversion of sugar mixtures into microbial products suchas ethanol. The key characteristic of this approach is the use ofmultiple strains which are each selective in their consumption of acarbon source. Excluding substrate consumption in a strain by genedeletions represents an innovative shift from the long-studied approachof constructing a “do-it-all” organism for the conversion of multiplesubstrates into a desired product.

There are two significant advantages that the process has for thesimultaneous conversion of sugar mixtures, as exemplified by xylose andglucose. Most importantly, as demonstrated by the fed-batch process(FIG. 5), the system adapts to fluctuations in the feed stream, i.e.cultures actually grow in concert with the feed composition. Since theyare each specialists, the strains can not adversely compete with eachother for the consumption of the substrates. Using a fed-batch processprevents sugar accumulation, and permits each strain to convert itstarget sugar at high yield and productivity. Operational robustness isthe hallmark of this process strategy, and it constitutes a majoradvance toward the utilization of lignocellulosic biomass. Second,although not part of this study, additional metabolic engineeringstrategies can focus on improving the individual production strainsindependently. For example, work can now be devoted to improving theglucose-selective strain for ethanol production with minimal concern forhow these changes would impact the conversion of xylose. We do not needto compromise one objective for another.

Gene knockouts affecting only one carbohydrate consumption pathwayappeared not to have deleteriously impacted the consumption of the othercarbohydrate. Indeed, previous results have demonstrated improved xyloseutilization in sugar mixtures by the ptsG knockout alone (Kimata et al.,1997, Proc Natl Acad Sci USA. 94:12914; Nichols et al., 2001, ApplMicrobiol Biotechnol. 56:120). In this study, catabolite repression dueto the presence of glucose was made irrelevant by the use of twostrains, since one cannot utilize glucose at all.

The results demonstrate that a population of substrate-selectivestrains, in which each individual strain only consumes a single sugar,is better able to metabolize a sugar mixture than a single strainconsuming multiple sugars. Other than the mutations involving substrateconsumption, the strains used for this study did not contain additionalmutations which would cause them to generate a product preferentially.The next step would be to use this approach with microbial strainsspecifically modified to accumulate a desired product such as ethanol.This approach could potentially be extended to construct additionalstrains capable of the exclusive consumption of other sugars (e.g.,arabinose) or inhibitors such as acetic acid and furfurals that arefrequently found in lignocellulosic hydrolysates.

Example 2 Consumption of Acetate by Escherichia Coli

E. coli readily consumes acetic acid as a sole carbon/energy source(El-Mansi et al., 2006, Curr. Opin. Microbiol. 9:173), but it generallywill not consume acetate in the presence of other substrates from whichthe cells can derive more energy. Of course, the presence of acetatediminishes the rates at which other substrates are consumed. However, E.coli can be forced to grow on acetate and prevented from consumingglucose or xylose (for example) by knocking out the genes which encodefor glucose and xylose consumption. We refer to such a strain as“acetate-selective” because of the three substrates, acetate is itsexclusive carbon nutrient. When exposed to a hydrolysate containingacetate, xylose and glucose, only the acetate will be consumed. Thisapproach is merely an extension of the substrate-selective conceptintroduced above for xylose and glucose mixtures.

We tested whether acetate could be selectively removed from a mixture ofxylose, glucose and acetate. We used E. coli MG1655 to generate ALS1060.MG1655 is a common wild-type strain (Jensen, 1993, J. Bacteria175:3401), and we verified that it grows aerobically with acetate as thesole carbon source at a growth rate of approximately 0.24 h⁻¹. ALS1060has four knockouts of genes coding for proteins involved in xylose andglucose utilization (these genes were described above): ptsG, manZ, glk,and xylA. These four mutations should prevent the consumption of eitherxylose or glucose by ALS1060, but have no known effect on acetatemetabolism.

We grew ALS1060 in a batch process using a medium containing anextremely high proportion of acetate: 10 g/L acetate, 10 g/L xylose and20 g/L glucose. In this case, ALS1060 consumed 10 g/L acetate at agrowth rate of 0.16 h⁻¹, but left xylose and glucose unconsumed evenafter 40 hours (FIG. 9). The significant lag phase observed in thisexperiment can be attributed to using the poor MG1655 strain and thesudden exposure to 10 g/L acetate. Acetate consumption depends on thecellular balance between the glyoxylate shunt and isocitratedehydrogenase (Holms, 1986, Curr Top Cell Regul 28:69), and different E.coli strains would be expected to behave quite differently. Ourselection of MG1655 was merely to provide evidence that the conceptwould work; this strain turned out not to be a good acetate-grower. Wehave recently found E. coli strains which consume acetate at a growthrate over 0.70 h⁻¹ with no lag, and will use these strains to developthe acetate-selective strain as described in the experimental approach.

In the preliminary experiments reported in Examples I and II we havebeen able to 1) remove acetate selectively from a mixture of xylose,glucose and acetate and 2) consume xylose and glucose simultaneously ina mixture of these two sugars. These steps can be linked together in atwo-stage process to generate a product like ethanol, as conceptualizedin FIG. 1 for an acetate/xylose/glucose mixture. After removal ofacetate in Stage 1, the remaining mixture is subsequently fermented in asecond process stage (Stage 2) to the desired product. Because knock-outstrains are very stable, the strategy can readily be extended to anynumber of substrates. For example, an arabinose-selective strain will beunable to consume xylose and glucose, etc.

Example 3 Two Stage Fermentation

Rather than try to develop a single organism to accomplish all theprocess design goals required for lignocellulosic conversion, our novelapproach uses multiple strains to do tasks efficiently andindependently. Note that no competition exists in the envisionedco-culture (in Stage 2, FIG. 1). Competition involves multiple speciescompeting for the same substrate. In this case, the strains each seekonly their specific substrate and, being otherwise the same, do notinterfere with each other. Thus, potential shortcoming from competitionin a “mixed-culture” bioprocess is avoided.

There are several significant advantages that the envisionedcontinuous/fed-batch process has for the elimination of acetic acid andthe simultaneous conversion of sugar mixtures, as exemplified by xyloseand glucose. First, because the acetate-selective strain cannot grow inthe absence of acetic acid, these cells will ultimately lyse in thesecond stage. By lysing, the cellular nutrients derived from acetateutilization are available to support growth of cells present in Stage 2.So, the inhibitor actually enhances product formation by enriching thehydrolysate with growth nutrients. It does not matter if the cellsconsume additional nutrients such as nitrogen, phosphorus, sulfur. Thesenutrients are conserved and become readily available to the organisms inthe second stage. The goal of the first stage is only to consume acetate(quickly) and not consume any sugars.

Secondly, metabolic engineering strategies can focus on improving theindividual production strains independently. For example, additionalwork can be devoted to improving the glucose-selective strain forethanol production without concern for the impact of these changes onthe conversion of xylose or on acetate tolerance/degradation. We do notneed to compromise one objective for another.

Finally, the system adapts to fluctuations in the feed stream; that is,cultures actually grow in concert with the feed composition. Forexample, the system would respond to an increase in acetateconcentration merely by increasing the cell density in Stage 1.Regardless of the perturbation of acetate in the feed (within a largerange, as long as the cells remain carbon limited), this inhibitor willbe completely removed in Stage 1. Similarly, as we have alreadyobserved, the system responds to an increase in the feed xyloseconcentration by increasing the cell density of the xylose-selectivestrain in Stage 2 (with no change in the cell density of any otherstrain). Using a fed-batch process would prevent sugar accumulation, andeach strain would convert its target sugar at high yield andproductivity. Operational robustness is the hallmark of this processstrategy, and it constitutes a major advance toward the utilization oflignocellulosic biomass.

Example 4 Concurrent Use of Sugars by Escherichia Coli

In Example 1 we demonstrated that the concept of substrate-selectiveuptake works. Using E. coli we demonstrated that a xylose-selectivestrain will consume only xylose and a glucose-selective strain consumesonly glucose in a mixture of these two sugars and without impacting eachother. Moreover, in Example 2 we demonstrated that acetate can beselectively removed from a mixture of acetate, glucose and xylose.

The strains used in these studies were not isogenic, not derived fromoptimally-growing strains and did not generate any product such asethanol. We will construct a series of substrate-selective strains anddemonstrate the two-step process shown in FIG. 1 on simulated and actualhydrolysates. Strains are constructed based on gene deletion and/orheterologous gene transformation, then their behavior in controlledbioreactors is examined. After initial examination in bioreactors,additional molecular biology may be necessary to further improve oroptimize the strain.

The following examples describe various aspects of the invention:

Example 4. Concurrent use of sugars by Escherichia coli

Example 5. Introduction of ethanol pathways into the strains

Example 6. Fermentation of simulated sugar mixture

Example 7. Consumption of acetate by Escherichia coli without sugardegradation

Example 8. Simulated and real hydrolysates

Example 9. Concurrent use of xylose and glucose by Lactic Acid Bacteria

Like many sugars, glucose must first be phosphorylated before it can befurther metabolized by E. coli. The principal route to phosphorylateglucose is by the phosphotransferase system (PTS). Because the PTSphosphorylates several sugars, some of the enzymes involved have broadspecificities and can phosphorylate more than one sugar (Postma et al.,1993, Microbiol Rev. 57:543). Glucose can be phosphorylated by twodifferent enzymes of the PTS, glucosephosphotransferase andmannosephosphotransferase encoded respectively by the ptsG and manZgenes (originally designated as gpt and mpt). Furthermore, glucose canbe phosphorylated by the enzyme glucokinase which is encoded by the glkgene (Curtis and Epstein, 1975, J. Bacteria 122(3):1189). Strains thatare unable to use glucose as a carbon source can be constructed byknocking out the genes encoding for glucosephosphotransferase (ptsG),mannosephosphotransferase (manZ) and glucokinase (glk).

Xylose is transported into E. coli by the xylose transport system andonce internalized must be isomerized to xylulose by xylose isomerase andphosphorylated to xylulose-phosphate by xylose kinase before it can bemetabolized (David and Weismeyer, 1970, Biochim. Biophys. Acta.201:497). Strains that are unable to utilize xylose as a carbon sourcecan be constructed by knocking out the gene encoding for xyloseisomerase (xylA).

Arabinose is another sugar found in biomass hydrolysates, accounting forabout 5% of the total sugar. The consumption of arabinose into E. coliis essentially analogous to the xylose uptake process. Arabinoseconsumption can be eliminated by knocking out the araA gene.

The hexose galactose is a component in lignocellulosic hydrolysate, butis commonly found at concentrations of 1% or less (Lee, 1997, Appl.Microbiol. Biotechnol. 65:56). Galactose uptake is mediated bygalactokinase encoded by the galK gene, and galactose uptake iseliminated in a galK mutant. Because the composition of galactose inhydrolysate is so low, eliminating galactose uptake may be unnecessary.

The presence of the hexose mannose in lignocellulosic hydrolysate varieswidely between 0-12% depending on the biomass (Lee, 1997, Appl.Microbiol. Biotechnol. 65:56). Mannose is phosphorylated exclusively bythe mannose-specific components of the PTS (e.g.,mannosephosphotransferase) encoded by manX; manY and manZ. Preventingglucose uptake by the triple mutations as described above (manZ) alsoprevents mannose consumption. Similarly, knocking out xylA and araA (andgalK) has no impact on mannose consumption. Using the strategy outlinedso far, we cannot construct a strain which consumes mannose but notglucose.

In order to consume all the sugars present in lignocellulosichydrolysates simultaneously, strains are constructed which are expectedto consume each one of these sugars alone. Thus, the following strainswill be constructed:

-   -   a) glucose-selective strain: mutations in araA galK xylA manZ    -   b) xylose-selective strain: mutations in ptsG manZ glk araA galK    -   c) arabinose-selective strain: mutations in ptsG manZ glk xylA        galK    -   d) galactose-selective strain: mutations in ptsG manZ glk araA        xylA    -   e) mannose-selective strain: mutations in araA galK xylA or araA        galK xylA ptsG glk (these strains will also consume glucose)

Our preliminary experiments (Examples I and II) were conducted in E.coli K12 or its non-isogenic derivatives, and included xylose andglucose, not the two other uniquely consumed sugars galactose andarabinose. The construction of a completely substrate-selective strainrequires knocking out all other uptake systems. So, for example, toconstruct a xylose-selective strain, we knock out the glk, ptsG, manZ,galK and araA genes in the E. coli B strain. The B strain has beenchosen for this project because it is a widely used prototrophicderivative of wild-type E. coli that is well characterized and growsrapidly in both defined and complex media. Furthermore, our experienceis that this strain grows very well on a variety of carbon sources(e.g., xylose, arabinose, etc.). Other research groups have also favoredderivatives of the B strain (Ingram et al., 1999, Biotechnol. Frog15:855; Tao et al., 2001, J. Bacteriol. 183(10):2979). The E. coli geneknockouts are constructed using the Keio collection of single-geneknockout mutants (Baba et al., 2006, Mol. Syst. Biol. 2:2006.0008). Thelambda Red recombination system (Yu et al., 2000, Proc Natl Acad Sci USA97:5978; Datsenko and Wanner, 2000, Proc Natl Acad Sci USA. 97:6640) mayalso be employed to construct the required gene knockouts. For, controlpurposes substrate-selective strains that we construct can be comparedto similar publicly available strains (such as CGSC5457 from the E. coliGenetic Stock Center that contains mutations in the gik, ptsG, and manZgenes and thus also can not consume glucose). The 4 substrate-selectiveB strains (one for each of glucose, xylose, arabinose and galactose) areexpected to be useful platforms for other researchers to developprocesses for the formation of various products from the five sugars(including mannose).

Additionally, using the same approach we will construct 4substrate-selective using the ethanologenic E. coli strain “KO11.” Theresult is expected to be eight substrate-selective strains (four eachderived from B and KO11).

Example 5 Introduction of Ethanol Production Pathways intoSugar-Selective E. Coli Cells

Decades of research have led to great improvements in strains of E. colithat accumulate ethanol, and the approach described herein can beadapted to current or future E. coli ethanologenic strains. The approachcan be adapted readily to strains of E. coli which generate any othercompound.

The sugar-selective KO11 strain of Example 4 is expected to beethanologenic, since KO11 is ethanologenic. To make the sugar-selectiveB strain of Example 4 ethanologenic, ethanol pathways will be introducedinto those strains. The four sugar-selective B strains will betransformed with pLOI308 (Ingram et al., 1987 , Appl. Environ.Microbiol. 53:2420; Ingram and Conway, 1988, Appl. Environ. Microbiol.54:397), one of the better characterized plasmids that elevates ethanolformation in E. coli. This plasmid uses the Zymomonas mobilis pyruvatedecarboxylase and alcohol dehydrogenase genes. In Zymomonas mobilis,pyruvate is converted by pyruvate decarboxylase to acetaldehyde, whichis subsequently converted to ethanol by alcohol dehydrogenase. Togetherthese enzymes comprise nearly 10% of the soluble protein of wild-type Z.mobilis cells grown with glucose as the substrate (Doelle et al., 1993,Crit. Rev. Biotechnol. 13:57), enabling this organism to maintain a highconversion efficiency to ethanol.

The result is expected to be two sets of ethanologenic, sugar-selectivestrains of E. coli (four each derived from B and KO11; 8 total).

Example 6 Fermentation of Simulated Sugar Mixtures

This example relates to the characterization of processes to metabolizesugar mixtures, ultimately in the presence of acetic acid (as describedbelow in a subsequent section). Initial studies will be performed onsynthetic mixtures of 2-5 sugars (selected from the list of mannose,galactose, glucose, xylose and arabinose), while subsequent studies willbe conducted on real hydrolysates supplemented with additional nutrientsas required (e.g., nitrogen and phosphate sources). These studies willuse the 8 strains generated from Example 5, with 4 strains present in aco-culture at the same time when all 5 sugars are present.

We will first establish the operating ranges for the processes through aseries of chemostat experiments. While not generally used in industry,chemostat experiments provide an extremely useful way of determining theparameters necessary to design a relevant process (for example, afed-batch process which is commonly used.) Like all fermentationexperiments, these studies will involve highly-instrumented bioreactorsin which feed-rate, temperature, pH, nutrient levels, oxygenation, etc.can be controlled. By studying a range of controlled growth rates, wewill determine for each strain the biomass yields, specific rates ofconsumption/production of dissolved and gaseous compounds, and themaintenance energy requirements resulting from the various geneticperturbations. Like any cells growing on different carbon-sources, thesestrains will have differing maximum growth rates. Note that in achemostat the microbial growth rate is determined by the nutrient feedrate (dilution rate), but the biomass concentration is determined by thelimiting nutrient concentration. These maximum growth rates will helpestablish the maximum feed-rate of an envisioned fed-batch process.Operating a biological process at the maximum growth rate does not ingeneral result in the maximum practical product formation rate for avariety of reasons such as oxygen requirement (for an aerobic process),heat duty, reduced biomass yield, genetic regulation of cells, etc. Wewill study the strains individually on single-substrate media, thensingle strains on dual-substrate media, then multiple strains ondual-substrate media. Drug resistances introduced into some strains willserve as selective markers to permit us to quantify the fraction of eachstrain comprising the total population.

In addition to the principal carbohydrates, major and minor fermentationproducts including ethanol, acetate, lactate, formate, succinate will beanalyzed. We anticipate that significant lactate and formate will begenerated for the B-derived ethanologenic strains, and in this case theymay be further modified by deleting the enzymes responsible for theseproducts: deleting the ldh gene which encodes lactate dehydrogenase willeliminate lactate from being produced while deleting the pfl gene whichencodes pyruvate formate lyase will eliminate formate. We have used thisstrategy to increase the fermentation yields of several products in E.coli (Tomar et al., 2003, Appl. Microbiol. Biotechnol. 62:76; Lee etal., 2004, Appl. Microbiol. Biotechnol. 65:56; Smith et al., 2006, Appl.Microbiol. Biotechnol. 28:1695; Zhu et al., 2007, Appl. Environ.Microbiol. 73:456).

The chemostat experiments will provide us with parameters to enable thestudy and implementation of a fed-batch process. We will feed in themixed-sugar stream in a carbon-limited fashion (that is, each one of thefour strains will be carbon limited for their sugar substrate). Duringan initial process “phase” cells will grow aerobically, while in asubsequent production phase, reduced oxygen availability will directmost of the carbon to the product ethanol. As long as the carbon-limitedfeed rate is lower than the capacity for carbohydrate uptake, the cellsshould respond to changes in the concentration of either substratemerely by increasing the biomass as we have observed in preliminarystudies. We will confirm this expectation by introducing a temporallyvarying stream of mixed sugar into the fermenter, and monitoring how thecomposition of the culture changes (including the population of eachstrain). The result of this portion of the research will be a completeand quantitative description of the fermentation of sugar mixtures toethanol by substrate-selective strains.

Example 7 Consumption of Acetate by Escherichia coli without SugarDegradation

A strain of E. coli is constructed which consumes acetate but not any ofthe five principal sugars in biomass hydrolysate as described in Example2. This process will require knockouts of all the sugar uptake systems.We have already demonstrated in Example 1 that this approach works forxylose and glucose, the two principal sugars in most lignocellulosichydrolysates.

The strain(s) used to make these knockouts are those that exhibit highgrowth rate and high biomass yield on acetate. Tolerance of the organismto high acetate concentration is not important (within a range) becausewe envision the process operating in fed-batch or continuous mode undercarbon (i.e., acetate) limitation. Under these circumstances theconcentration of acetate in the fermenter will be maintained at zero andcell “tolerance” to the acetate will not be relevant. However, the rateat which acetate is consumed will directly affect the productivity ofthe entire process. Therefore, the cells should ideally be able to growat a high growth rate which will enable the process to run at a high(dilution) rate without any negative consequences.

Although several research groups have studied growth of E. coli onacetate and completed detailed flux analyses, there has not been acomprehensive comparison of the growth rate of E. coli strains onacetate as the sole carbon source. We will examine 8-10 diversewild-type and common strains of E. coli (e.g., MG1655, DH5α, MC4100,BL21, JM109, etc.) and grow them as accelerostats (Paalme et al., 1997,Ant. van Leeuwen. 71:217). B strains are preferred because these strainsappear to have an elevated expression of enzymes in the glyoxylate shunt(van de Walle and Shiloach, 1998, Biotechnol. Bioeng. 57:71; Phue andShiloach, 1998, J. Biotechnol. 109:21), which is an important pathwayfor acetate metabolism. A preliminary study in shake flasks hasindicated some strains do indeed grow with a very high rate on acetate(0.70 h⁻¹, about 3 times faster than on MG1655 which was selected forpreliminary studies). In this study we determined the growth rate ofseveral publicly available bacterial strains. The table below showsobserved growth rates.

Specific Growth biomass Rate yield Strain (h-1) (g/g) AG1 0.676 0.303MC1061 0.520 0.315 MC4100 0.371 0.279 9637 0.368 0.327 JM101 0.322 0.285BL21 0.305 0.232 MG1655 0.253 0.309 W3110 0.245 0.281This approach can be readily used establish biomass yield and maximumgrowth rate. The two “best” strains, AG1 and MC1061, are selected toknockout the sugar-consuming abilities (described below). Note that thestrain found to be the “best acetate consumer” will not necessarily berelated to the strain ultimately found to be the “best sugarconsumer/ethanol producer.” A significant advantage of our processdesign is that these two strains can be selected independently.

In order to learn why certain strains grow more quickly on acetate, wewill complete a genome-wide microarray study. For four of the strains(two “fast growers” and two “slow” growers), we will take samples fromour accelerostat experiments (which occurs at a pseudo-steady state) atthree different growth rates (approx. 0.1, 0.2, 0.3 h⁻¹). We willconduct microarrays comparing expression at these growth rates (i.e.,0.3 vs. 0.1 and 0.2 vs. 0.1) and at the highest growth rate between thestrains (strain 2 vs. strain 1, strain 3 vs. strain 1, strain 4 vs.strain 1). With (independent) triplicate experiments, this will involve15 microarrays. This approach is similar to our previous study at 6steady-state growth rates to establish the regulatory importance of thearcAB regulatory network in acetate overflow metabolism (Vemuri et al.,2006, Appl. Environ. Microbiol. 72(5):3653).

Once suitable strains of E. coli are identified, they must be madeacetate-selective, i.e., they will need deletions in genes involved inthe uptake of glucose, mannose, galactose, xylose and arabinose. In thetwo strains selected for their “best” acetate metabolism, the six genesmanZ, ptsG, glk, xylA, galK, araA will be knocked out as described inExample 4. None of these genes has any known relationship with acetateconsumption, a process which is mediated by acetyl CoA synthase (acs),isocitrate lyase (aceA), malate synthase (aceB) and isocitratedehydrogenase (icdA) (Holms, 1986, Curr Top Cell Regul 28:69).

Example 8 Use of Simulated and Real Hydrolysates

Two-stage fermentations of simulated mixed xylose, glucose, mannose,arabinose, galactose and acetate solutions as well as actualhydrolysates will be conducted using the constructed E. coli strains. A“simulated” solution is merely a synthetic (and reproducible) mediumprepared with purified components in appropriate proportions torepresent a real hydrolysate. In other words, we will prepare a definedmedium containing these six compounds as potential carbon sources.Concentrations of these compounds in actual hydrolysates varyconsiderably (Barbosa et al., 1992, Appl. Environ. Microbiol. 58:1382;Johansson et al., 2001, Appl Environ Microbiol. 67:4249; Taherzadeh etal., 2001, Appl Biochem Biotechnol. 95:45; Brandberg et al., 2004, J.Biosci Bioeng. 98:122), and we will examine a range of concentrations:20-40 g/L glucose, 5-20 g/L xylose, 1-5 g/L galactose/mannose/arabinoseand 2-8 g/L acetic acid. Although we have the ability to generatebiomass hydrolysates, we will primarily rely on real hydrolysatesobtained from researchers at other Universities and Federal Labs. Asnecessary, the hydrolysate will be supplemented with other nutrientssuch as nitrogen, phosphorus and sulfur.

The initial studies will use simulated hydrolysates. The first stage inthe process (the left bioreactor in FIG. 1) involves the removal ofacetic acid by feeding an acetate-containing hydrolysate into thereactor so that the organisms grow continuously and the acetate-freestream with cells exits the vessel continuously. Using the growth ratedata obtained previously, we will feed the acetate at a dilution ratebelow the maximum growth rate and scale the process accordingly. We willdetermine the ranges of acetate concentration in the hydrolysate thatare acceptable, and the rates for which the process can be conducted. Wewill determine the long-term stability of the process. The acetateconsumption rate determined from such data will be helpful in sizing apilot/commercial process. When acetate is the limiting nutrient underaerobic conditions, we anticipate that the carbon in acetate will beconverted either into cells or CO₂. We will nevertheless need tocomplete a detailed carbon balance to account for the utilization ofcarbon under various operating conditions. The formation of thepotential metabolic products such as ethanol, lactate, formate,succinate, and fumarate in the detoxification step is not anticipatedbut will be determined in addition to the concentrations of the sixsubstrates via chromatography (Eiteman and Chastain, 1997, Anal. Chim.Acta 338:69).

Additionally, we will demonstrate the robustness of the process by“ramping” the concentration of acetate in the feed and observing theresponse of the microbial system to changing acetate concentration. Weanticipate that the cells will naturally adapt to changingconcentrations of acetate in the feed stream by increasing the biomassconcentration, as we have observed in preliminary studies for thetwo-substrate system. For simulated hydrolysate we anticipate that thestream exiting Stage 1 will contain exclusively 5 sugars (and biomass)as carbon sources.

Stage 2 will be fed the stream exiting Stage 1. It is advantageous tomaintain Stage 1 as a continuous (or fed-batch) process to maintain azero acetate concentration as a result of substrate limitation. Thematching of this continuous process with Stage 2 will be a significantaspect of this part of the research project. Three different bioprocessmodes will be examined for Stage 2, including: 1) batch, 2) linearfed-batch, and 3) exponential fed-batch. A batch process will beconducted by fermenting a discrete portion of the (continuous) effluentfrom Stage 1. Such a process will require storing some of the Stage 1effluent in a tank. A linear fed-batch will be conducted bysynchronizing the Stage 1 effluent rate with the feeding rate to Stage2. In this case, because the biomass concentration in Stage 2 is smallthe feed initially into Stage 2 will exceed the rate of carbohydrateconsumption, and the cells will grow at their maximum growth rate. Laterafter the biomass concentration increases, the maximum growth rate willexceed the carbohydrate feed rate and Stage 2 will become carbonlimited. An exponential fed-batch, accomplished via a programmable pump,will involve holding a portion of the Stage 1 effluent initially andgradually increasing the rate of feeding to match a desired cell growthrate. We routinely perform exponential fed-batch processes (e.g., Smithet al., 2006, Appl. Microbiol. Biotechnol. 28:1695) as they areadvantageous to control growth and product formation rates carefully.The focus of this portion of the research will be to study the kineticsof sugar consumption subsequent to acetate removal. Process stability,mode of operation, robustness to varying feed compositions andsensitivity to inoculation approaches will all be addressed in thisportion of the project.

We will study ethanol production in this process, using a singleorganism process with E. coli KO11 (without additionalsubstrate-selectivity) as an experimental control. Although a couple ofdifferent ethanol-production approaches are possible depending on thespecific strain, we envision a two-step process for Stage 2 wherein thefirst step is an aerobic growth phase using the two strains andsubstrates. A second step involves a potential anaerobic productionphase wherein all the carbon is directed to the product of interest,ethanol, and growth is low. Growth can be slowed by several meansincluding limiting the feed in another nutrient such as nitrogen (i.e.,ammonium ion).

We will also obtain lignocellulosic hydrolysates and determine thedetoxification of that hydrolysate using the acetate-selective strain inthe developed process. As noted, it is likely that the raw hydrolysatewill require supplementation with additional nutrients in order topermit growth of the acetate-selective strain in Stage 1 and the growthof the 4 sugar-selective strains in Stage 2. Several commercialprocesses use nutrient supplementation which does not negatively impactthe cost of production (ammonia, phosphate). We will readily be able todetermine whether the hydrolysate is, for example, phosphate-limitedrather than carbon-limited by determining the nutrient levels in theeffluent stream. We anticipate that the feasible feed rates for bothstages will be lower when lignocellulosic hydrolysate is used comparedto simulated hydrolysate. The results from these studies will becritically compared with the results obtained using the simulatedxylose, glucose and acetate solution.

Example 9 Concurrent Use of Xylose and Glucose by Lactic Acid Bacteria

So-called facultative homofermentative strains of Lactic Acid Bacteriaare of great interest because they are able to consume either xylose orglucose (and other sugars), and many also have a high tolerance to lowpH and temperatures exceeding 50° C. These qualities make thesemicroorganisms particularly relevant for the production of lactic acid(currently manufactured commercially) and other products includingethanol. The substrate-selective approach of the invention can beadvantageously utilized in this organism. Strains of Lactobacillus andLactococcus which are able to consume selectively xylose and glucosewill be constructed. Other researchers have explored ethanol productionin Lactobacillus (Nichols et al., 2003, J. Indust. Microbiol.Biotechnol. 30:315). This study is expected to show that (1)inhibitor-removal and sugar-conversion steps are decoupled in the methodof the invention, and (2) two different species can be used in the twoseparate stages without any disadvantages. Importantly, because of theseadvantages, researchers no longer have to pass over a strain for theproduction of a biochemical merely because it is intolerant of anotherchemical which could now be removed (using our approach) in a previousstage by another organism.

First, isogenic xylose-selective and glucose-selective strains of LacticAcid Bacteria will be constructed. A Lactic Acid Bacteria strain thatcan consume both xylose and glucose is chosen as the starting strain fordeveloping the isogenic xylose-selective and glucose-selective strains.Like E. coli, Lactic Acid Bacteria possess glucose-specific uptakepathways in addition to the general PTS which facilitates the import ofa variety of sugars. The cytoplasmic phosphocarrier enzymes of the PTShave been identified in several Lactic Acid Bacteria species and areencoded by the ptsH and ptsI genes which reside in an operon, and as inE. coli, Lactic Acid Bacteria ptsHI deletion mutants are still able tometabolize glucose (Stentz et al., 1997, Appl. Environ. Microbiol.63:2111; Luesink et al., 1999, J. Bacteriol. 181:764; Viana e al., 2000,Mol. Microbiol. 36:570). However, because the glucokinase homologue hasnot been identified in a Lactic Acid Bacteria species in which the ptsHIoperon has also been characterized, we will start with a Lactic AcidBacteria species in which ptsHI has been deleted in order to construct axylose-selective Lactic Acid Bacteria strain.

Lactobacillus casei, Lactobacillus sake, and Lactococcus lactis arespecies of Lactic Acid Bacteria in which the ptsHI genes have beencharacterized and which also can utilize xylose as a sugar source (Zhanget al., 1995, Appl. Biochem. Biotechnol. 51/52:527; Erlandson et al.,2000, Appl Environ Microbiol. 66:3974). Similar to the E. coli studiesin which we select a strain for acetate consumption (Example 7), we willfirst compare these three candidate Lactic Acid Bacteria to determinetheir growth rates in minimal xylose media to choose a preferred strainfor these studies. ptsHI derivatives of each of these three strains areavailable (Stentz et al., 1997, Appi. Environ. Microbiol. 63:2111;Luesink et al., 1999, J. Bacteriol. 181:764; Viana e al., 2000, Mol.Microbiol. 36:570), and a ptsHI deletion derivative of the selectedstrain will be mutagenized in order to generate a Lactic Acid Bacteriathat is incapable of growing on minimal glucose media. Bothnitrosoguanidine and ethyl methanesulfonate will be employed as mutagensand if necessary antibiotic enrichment procedures and MacConkey Glucoseselective plates will be used to identify a Lactic Acid Bacteria ptsHImutant that is xylose-selective and incapable of metabolizing glucose.

An isogenic glucose-selective Lactic Acid Bacteria strain will then begenerated. Like E. coli, Lactic Acid Bacteria strains which containmutations in either the genes that encode for the xylose transporter orisomerase or xylulokinase enzymes cannot utilize xylose as a sole carbonsource (Lokman et al., 1991, Mol Gen Genet. 230:161; Chaillou et al.,1998, Appl. Environ. Microbiol. 64:4720; Erlandson et al., 2000, ApplEnviron Microbiol. 66:3974). Chemical mutagenesis will be employed togenerate a Lactic Acid Bacteria xyl mutant using the same “optimal”strain as described above. The result will be two isogenic strains of aspecies of Lactic Acid Bacteria, one which is xylose-selective and asecond which is glucose-selective.

Next, fermentations of sugar mixtures will be conducted. Analogous tothe more comprehensive research completed for E. coli in the aboveExamples, as part of this experiment with Lactic Acid Bacteria, we willcomplete fermentations of a medium composed of xylose and glucose usingthe substrate-selective strains. A unique aspect of many Lactic AcidBacteria is their tendency to switch between heterofermentative andhomofermentative conditions as a function of growth rate and redoxconditions (Garrigues et al., 1997, J. Bacteria 179:5282). We will studyboth low growth rates at which mixed-acid (and ethanol) production isexpected and high growth rates at which lactic acid productiondominates. Previous difficulties with ethanol production in Lactic AcidBacteria (Nichols et al., 2003, J. Indust. Microbiol. Biotechnol.30:315) may be attributed to the competition between lactic acidformation and ethanol formation under high growth, homofermentativeconditions. Although the focus will be the simultaneous utilization ofxylose and glucose and not on product formation, per se, anunderstanding of product formation and its relationship to operationalparameters is important in the interpretation and analysis of theresults.

We will conduct chemostat experiments to establish the operating rangesfor the processes and the performance under both homofermentative andheterofermentative conditions. We will determine for each strain thebiomass yields, specific rates of consumption/production of dissolvedand gaseous compounds, and the maintenance energy requirements resultingfrom the various genetic perturbations. We will study the strainsindividually on single-substrate media, then single strains ondual-substrate media, then multiple strains on dual-substrate media. Wewill assess whether the presence of the unutilized sugar impacts theability of the strain to consume its exclusive substrate, determine theoperating optimal conditions and ranges for the conversion process, andexamine how the distribution of end-products is affected by theco-culture. These experiments will facilitate the development of futureprocesses to convert lignocellulosic hydrolysates efficiently intouseful products with other organisms such as yeast.

Example 10 Substrate-Selective Saccharomyces Cerevisiae

Because yields and productivity are the two most important economicfactors in the manufacture of fuel ethanol, the preferred microorganismof choice is Saccharomyces cerevisiae. This yeast ferments ethanol veryefficiently and can tolerate the highest ethanol concentrations of anyknown microorganism. Unfortunately, the use of S. cerevisiae to producefuel ethanol from lignocellulosic hydrolysates is problematic, becauseit cannot utilize the pentose sugars xylose and arabinose which are twoof the most abundant sugars that are found in lignocellulosichydrolysates. Researchers have attempted to solve this problem byconstructing new S. cerevisiae derivatives that contain the genesrequired for xylose utilization. These new yeast derivatives can utilizexylose, however, when these yeasts are fed a mixed carbon source thatcontain glucose and xylose, the glucose must be consumed first, beforeany xylose can be utilized.

Our multiple cell approach can be readily applied to S. cerevisiae. S.cerevisiae strains that cannot utilize galactose can be constructed bydeleting the GAL2, GAL1, GAL7, or GAL10 genes. These strains will beable to utilize glucose as a carbon source. S. cerevisiae strains thatcannot utilize glucose can be constructed by deleting the GLK1, HXK1,and HXK2 genes or the HXT1, HXT2, HXT3, HXT4, HXT6, HXT7, and SNF3genes. These strains will be able to utilize galactose as a carbonsource. S. cerevisiae strains that can utilize xylose or arabinose canbe constructed by importing these pathways from other yeasts such asPichia stipitis or Ambrosiozyma monospora which are able to utilizethese sugars. By knocking out the genes that are required for glucoseand galactose utilization in S. cerevisiae strains that have beengenetically modified to consume xylose and/or arabinose, new strains canbe created which are only able to utilize xylose and/or arabinose.Concurrent use of a combination of these strains would be able toproduce ethanol very efficiently.

Example 11 A Substrate-Selective Co-Fermentation Strategy withEscherichia coli Produces Lactate by Simultaneously Consuming Xylose andGlucose

We report a new approach for the simultaneous conversion of xylose andglucose sugar mixtures which potentially could be used forlignocellulosic biomass hydrolysate (Eiteman et al., 2009 Biotechnol.Bioeng. 201:822-827). We used this approach to demonstrate theproduction of lactic acid. This process uses two substrate-selectivestrains of Escherichia coli, one which is unable to consume glucose andone which is unable to consume xylose. In addition to knockouts in pflBencoding for pyruvate formate lyase, the xylose-selective (glucosedeficient) strain E. coli ALS1073 has deletions of the glk, ptsG andmanZ genes while the glucose-selective (xylose deficient) strain E. coliALS1074 has a xylA deletion. By combining these two strains in a singleprocess the xylose and glucose in a mixed sugar solution aresimultaneously converted to lactate. Furthermore, the biomassconcentrations of each strain can readily be adjusted in order tooptimize the overall product formation. This approach to the utilizationof mixed sugars eliminates the problem of diauxic growth, and providesgreat operational flexibility.

Recently, we proposed a multi-organism approach to utilizing a sugarmixture (Eiteman et al., 2008, J Biol Eng 2:3). This approach involvesintroducing into a mixed substrate stream several strains which each areable to consume only one particular substrate. Each strain willtherefore effectively ignore other substrates while it carries out theone target conversion. An advantage of such “substrate-selective uptake”is that the system can adapt to fluctuations in the feed stream; thatis, cultures can grow in concert with a variable feed composition. Also,metabolic engineering strategies could ultimately focus on improving theindividual production strains independently. For example, theglucose-selective strain could be improved for the generation of aparticular product without having to compromise on how those changesmight impact the conversion of xylose to that product.

In this example we use substrate-selective strains of Escherichia colifor the formation of a product of interest, such as lactic acid, in amixed-sugar defined medium. Lactate has previously served as aconvenient product to show conversion of xylose and glucose mixturesusing a ptsG mutant of E. coli in complex medium (Dien et al., 2002, JIndustr Microbiol 29:221-227). Over 1.5M lactate can be generated bythis organism having several key mutations under carefully controlledconditions (Zhu et al., 2007, Appl Environ Microbiol 73:456-464). In thepresent study, we use metabolically engineered strains to study thefeasibility of a two-strain strategy for the simultaneous conversion ofxylose and glucose into lactate.

Methods Strains

The Escherichia coli strains used in this study are listed in thefollowing table. Deletion mutants from the Keio collection (Baba et al.,2006, Mol Syst Biol. 2:1-11) were moved into strains by P1 transduction,and Kan(R) was subsequently deleted using the curable pCP20 plasmidwhich overproduces Flp recombinase (Cherepanov and Wackernagel, 1995,Gene 158:9-14). The Δ(pflB::Cam) deletion was moved into strains by P1transduction. Using aerobic shake flask cultures, we verified thatALS1073 would not consume glucose, while ALS1074 would not consumexylose.

Strain Genotype Source JW1087-2 Δ(araD-araB)567 ΔlacZ4787(::rrnB-3) 1Δ(rhaD-rhaB)568 hsdR514 rph-1 λ-ΔptsG763::(FRT)Kan JW1808-1Δ(araD-araB)567 ΔlacZ4787(::rrnB-3) 1 Δ(rhaD-rhaB)568 hsdR514 rph-1λ-ΔmanZ743::(FRT)Kan JW2385-1 Δ(araD-araB)567 ΔlacZ4787(::rrnB-3) 1Δ(rhaD-rhaB)568 hsdR514 rph-1 λ-Δglk-726::(FRT)Kan JW3537-1Δ(araD-araB)567 ΔlacZ4787(::rrnB-3) 1 Δ(rhaD-rhaB)568 hsdR514 rph-1λ-ΔxylA748::(FRT)Kan MG1655 F− λ- 2 NZN111 F+ λ-rpoS396(Am) rph-1ldhA::Kan ΔpflB:Cam 3 ALS1038 F− λ-ΔxylA748::FRT 4 ALS1048 F−λ-ΔptsG763::FRT ΔmanZ743::FRT 4 Δglk-726::FRT ALS1073 F− λ-ΔpflB::CamΔptsG763::FRT ΔmanZ743::FRT 4 Δglk-726::FRT ALS1074 F− λ-ΔpflB::CamΔxylA748::FRT 4 1) Baba et al., 2006, Mol Syst Biol. 2: 1-11. 2) Guyeret al., 1980, Cold Spring Harbor Symp Quant Biol 45: 135-140 3) Bunch etal., 1997, Microbiology 143: 187-195. 4) This example

Growth Conditions

Basal medium contained (per L): 13.3 g KH₂PO₄, 4.0 g (NH₄)₂HPO₄, 1.2 gMgSO₄.7H₂O, 13.0 mg Zn(CH₃COO)₂.2H₂O, 1.5 mg CuCl₂.2H₂O, 15.0 mgMnCl₂.4H₂O, 2.5 mg CoCl₂.6H₂O, 3.0 mg H₃BO₃, 2.5 mg Na₂MoO₄.2H₂O, 100 mgFe(III)citrate, 8.4 mg Na₂EDTA.2H₂O, 1.7 g citric acid, and 4.5 mgthiamine.HC1. General “BXG” medium contained basal medium plus xyloseplus glucose. Two different compositions of xylose and glucose were usedin this study, “B2X3G” medium comprised basal medium with nominally 20g/L xylose and 30 g/L glucose, while B3X2G contained 30 g/L xylose and20 g/L glucose. For each bioreactor experiment, a single strain wasfirst grown in a tube containing 10 mL BXG medium, then 5 mL transferredto 50 mL BXG medium in a 250 mL shake flask. All flasks were incubatedat 37° C. and 250 rpm (19 mm pitch), and the pH was adjusted to 6.7 with20% NaOH. For those fermentations in which a single strain was used,when the OD of the shake flask culture reached approximately 4, theflask contents were diluted with BXG medium so that 100 mL having aneffective OD of 1.0 was used to inoculate the bioreactor. For thoseexperiments in which two strains were used in a single process, thecontents of two shake flasks were diluted with BXG medium to 100 mL sothat each strain had an effective OD of 1.0 (i.e., in the 100 mLvolume). Identical “BXG” media were used for a given experimentalsequence consisting of tube culture, shake flask culture and bioreactorculture.

Fermentation

Batch experiments were carried out in a 2.5 L bioreactor (Bioflo 2000,New Brunswick Scientific Co. Edison, N.J., USA) containing 1.0 L BXGmedium. Each experiment consisted of two process phases maintained at37° C. During an initial aerobic growth phase with duration as reportedin the results, air was sparged into the fermenter at a flowrate of 1.0L/min, and the agitation was 1000 rpm to ensure no oxygen limitation.The pH was controlled at 6.7 using 28% (w/v) NH₄OH. During the secondanaerobic phase, N₂ was supplied at 0.2 L/min, the agitation was reducedto 200 rpm, and the pH was controlled at 6.7 using 20% (w/v) NaOH. Theproduct yields reported are the amount of product formed divided by theamount of sugar consumed during the second production phase.

Analyses

The optical density at 600 nm (OD) (DU-650 spectrophotometer, BeckmanInstruments, San Jose, Calif.) was used to monitor cell growth, and thisvalue was correlated to dry cell mass. Previously described liquidchromatography methods were used to quantify organic compounds (Eitemanand Chastain, 1997, Anal Chem Acta 338:69-75).

Results and Discussion

Escherichia coli ALS1074 has a knockout in the xylA gene encoding forxylose isomerase, rendering ALS1074 unable to consume xylose. Thexylose-selective strain ALS1073 has mutations in the three genesinvolved in glucose uptake (Curtis and Epstein, 1975, J Bacteriol122:1189-1199), rendering it unable to consume glucose: ptsG encodes theEnzyme IICB^(Glc) of the phosphotransferase system (PTS) forcarbohydrate transport (Postma et al., 1993, Microbiol Rev 57:543-594),manZ encodes the IID^(Man) domain of the mannose PTS permease (Huber andErni, 1996, Eur J Biochem 239:810-817), glk encodes glucokinase (Curtisand Epstein, 1975, J Bacteriol 122:1189-1199). Each strain also has amutation in pflB encoding pyruvate formate lyase, which causes a severemetabolic bottleneck at pyruvate under anaerobic conditions, curtailinggrowth in the absence of acetate and diverting most carbon to lactate(de Graef et al., 1999, J Bacteriol 181:2351-2357).

In our first experiment, we grew ALS1073 or ALS1074 (i.e., each strainalone) under aerobic conditions in B2X3G medium, and after 8 h switchedto anaerobic conditions. Sugar concentrations were used whichrepresented the levels found in most lignocellulosic hydrolysates.ALS1074 consumed approximately 10 g/L glucose during the 8 h of growthto an OD of 11.5 (FIG. 10). After initiating anaerobic conditions,growth stopped and the remaining 17 g/L glucose was converted into about14 g/L lactate within 3 h for a yield of 0.83 g/g (based on substrateconsumed during the anaerobic production phase only). About 1.5 g/Lsuccinate and less than 0.5 g/L acetate and ethanol as by-products werealso generated during the anaerobic phase (yields: 0.095 g succinate/gglucose, 0.004 g acetate/g glucose, 0.028 g ethanol/g glucose). Lactatewas formed at a constant specific rate of 1.2 g/g·h during the anaerobicphase, and throughout the process the xylose concentration remainedunchanged.

ALS1073 consumed 4 g/L xylose during the 8 h aerobic growth phase to anOD of 4.5 (FIG. 11). During the anaerobic phase, the remaining 13.3 g/Lxylose was converted to 13 g/L lactate at a yield near 1.0 g/g. Theconversion of xylose to lactate was completed in 14 h. The rate ofxylose consumption during the anaerobic phase by ALS1073 appears to bemuch slower than the rate of glucose consumption by ALS1074 (shown inFIG. 10). However, the biomass concentration was appreciably differentbetween the two experiments—the biomass concentration of ALS1073 after 8h of growth on xylose was less than 40% of the biomass concentrationusing ALS1074 on glucose. The specific rate of xylose consumption was0.92 g/g·h at the onset of the anaerobic phase, whereas the rate ofxylose consumption was 0.49 g/g·h during the latter portion of theanaerobic phase. Less than 0.5 g/L succinate (0.023 g succinate/gxylose) and no acetate and ethanol were generated as by-products, andthe glucose concentration remained unchanged during the entire process.

In a second experiment, both ALS1073 and ALS1074 were inoculated into asingle bioreactor containing the same xylose-glucose defined mediumB2X3G. In this two-strain co-fermentation, care was taken to ensure thateach strain was inoculated at approximately the same cell density thatwas used in the two different one strain processes. After 8 h of growththe anaerobic phase was similarly initiated, and at this time theculture had consumed about 4 g/L xylose and 10 g/L glucose to achieve anOD of 16 (FIG. 12). During the anaerobic phase, the remaining glucosewas consumed in less than 3 h, and the xylose was consumed in about 12h. Assuming that the measured OD of 16 represents an OD of 11.5 forALS1074 and an OD of 4.5 for ALS1073 (values observed in the previoussingle organism cases), then the specific rate of glucose consumptionduring the anaerobic phase was 1.6 g/g·h, and the rate of xyloseconsumption was initially 1.2 g/g·h. The 17.5 g/L glucose and 13.3 g/Lxylose present at the onset of the anaerobic phase were converted to25.9 g/L lactate, for a yield of 0.84 g lactate/g total sugar. The sameminor by-products were also generated, with yields based onxylose+glucose of 0.063 g succinate/g, 0.008 g acetate/g, 0.014 gethanol/g. Interestingly, the final by-product concentrations wereapproximately the sum of what was observed in the twosingle-substrate-consumption experiments. For example, the finalsuccinate concentration when two strains converted two sugars was 1.94g/L, which is approximately the sum of the concentrations observed inthe xylose-only (0.30 g/L) and glucose-only (1.62 g/L) experiments. Thisexperiment demonstrates that the two strains acted independently in theconversion of xylose and glucose to lactate.

Although the two-strain process as implemented (FIG. 12) performedexactly as each single strain process, the two-strain process exposedone important shortcoming. Under the conditions of the experiment, thevolumetric rate of xylose consumption did not match the volumetric rateof glucose consumption. Specifically, because glucose exhaustionoccurred in less than 3 h of anaerobic conditions but xylose consumptionrequired over 12 hours, the process inefficiently consumed only one oftwo possible substrates for the final 10 h. The overall process wasessentially limited by the volumetric rate of xylose consumption. Tomaximize overall productivity, the two consumption rates ideally wouldallow both glucose and xylose to become exhausted at about the sametime. Moreover, these two consumption rates should be adjustable so thatthis optimal productivity occurs regardless of the initialconcentrations of xylose and glucose.

The volumetric consumption rate (Q) is equal to the specific consumptionrate (q) times the biomass concentration (X). So, for thexylose-selective strain Q_(xtlose)=q_(xylose)X_(xylose). One generalmethod to increase Q is to increase q, for example using metabolicengineering approaches directed toward altering the pathways involvingthat substrate. Another general method to increase Q is to increase thebiomass concentration of the strain. Because q_(glucose) is about 30%greater than q_(xylose) during the anaerobic phase for the particulartwo-strain bioprocess we conducted, a higher cell density for thexylose-selective strain X_(xylose) is needed relative to X_(glucose) inorder to match the two values of Q. In a one-strain approach for thesimultaneous consumption of xylose and glucose, only one biomassconcentration exists, and the absence of this additional degree offlexibility prevents the matching of utilization rates for multiplesubstrates.

To illustrate the flexibility in aligning consumption rates, we repeatedthe two-strain experiment using the same B2X3G medium. In this case, weincreased the cell density of the xylose-consuming strain by providingthis strain more time for growth prior to switching to the non-growthproduction phase. Specifically, using a nearly identical medium with 20g/L xylose and 31 g/L glucose, at the start of the process (t=0) thebioreactor was only inoculated with the xylose-consuming strain ALS1073.Two hours after this inoculation, the bioreactor was inoculated with theglucose-consuming strain ALS1074, and at 8.5 h, anaerobic conditionscommenced. Therefore, the xylose-consuming strain experienced 8.5 h ofaerobic growth, while the glucose-consuming strain was allowed only 6.5h of aerobic growth. At the time that anaerobic conditions commenced,the OD of the culture was approximately 10.5 with 27.6 g/L glucose and15.4 g/L xylose (FIG. 13), and we estimate that about 60% of the biomasswas ALS1073 while 40% of the biomass was ALS1074. In this experiment,the rates of glucose and xylose consumption were much more closelymatched, and both sugars were consumed almost simultaneously. Thus, thetwo-sugar mixture was efficiently converted at a constant rate into 32g/L lactate over the course of 8 h. We also observed about 2.5 g/Lsuccinate and 0.5 g/L ethanol and acetate (yields based onxylose+glucose: 0.84 g lactate/g, 0.065 g succinate/g, 0.004 gacetate/g, 0.012 g ethanol/g). The small sacrifice made in theunnecessarily large glucose consumption rate was more than offset by theimprovement in the xylose-consumption rate.

This particular method can be used to tailor the volumetric consumptionrates for other concentrations of xylose and glucose. To demonstratethis flexibility, we conducted a bioprocess using a medium withinitially 33 g/L xylose and 22 g/L glucose (nominally B3X2G). Becausethis medium contains 50% more xylose than glucose, an even higher celldensity of the xylose-selective strain ALS1073 relative to theglucose-selective strain ALS1074 is required compared to the previousprocess using B2X3G medium. Therefore, at the start of the process (t=0)the culture was inoculated with ALS1073, while the inoculation withALS1074 occurred 3.1 h later. Anaerobic conditions commenced at 9.1 h;thus, ALS1073 experienced 9.1 h of growth while ALS1074 experienced 6 hof growth. At the onset of anaerobic conditions, the OD of the culturewas approximately 12 with 19.5 g/L glucose and 20.4 g/L xylose (FIG.14). Again, the rates of glucose and xylose consumption were closelymatched, and the two-sugar mixture was efficiently converted into 37 g/Llactate with a lactate-sugar yield of 0.88 g/g. Other products generatedduring anaerobic conditions included 0.069 g succinate/g, 0.010 gacetate/g, 0.009 g ethanol/g.

Conclusions

The process described in this study demonstrates the simultaneousconversion of sugar mixtures into one particular product, lactate. Inthis case, two strains were used which are each selective in theirconsumption of the two carbon sources present, xylose and glucose.Excluding substrate consumption in a strain by gene deletions representsa new approach for the conversion of multiple substrates into a desiredproduct. If additional carbon sources were present, then additionalstrains could be constructed to ensure each strain exclusively convertedone substrate into the desired product. One advantage demonstrated isthat not only can xylose and glucose be simultaneously converted to aproduct like lactate, but the conversion rates of each sugar can beindividually modulated to optimize the overall process. In this study,this “alignment” of consumption rates was exacted by inoculating theculture at different times, thereby allowing each strain to reach adesired cell density prior to switching to a non-growth productionphase. However, other means might be available to align consumptionrates in other circumstances, for example, using differential inoculadensities or introducing genetic modifications which affect growthrates. The multi-strain approach therefore offers a significantadvantage over any one-strain approach to mixed sugar utilization.Presumably another advantage, not explored in this study, is that eachstrain can be independently engineered genetically to maximize onesugar-to-product conversion.

Example 12 Acetate Consumption by Pseudomonas Spp

We studied the exclusive consumption of acetate by E. coli; specificallywe compared numerous E. coli strains to learn which strains had thehighest rate of acetate consumption. We found that most E. coli strainshave growth rates of about 0.25 h⁻¹ on acetate, whereas the fastest E.coli strain showed a growth rate of about 0.35 h⁻¹. Because we areinterested in developing a strain that consumes acetate as quickly aspossible, we also tested several Pseudomonas species.

Like E. coli, most Pseudomonas are prototrophic, do not require aminoacids supplements, and grow very quickly on minimal defined media. Wetested three different strains of Pseudomonas and two of them had growthrates of about 0.70 h⁻¹ on acetate. This growth rate is about 3 timesfaster than an average E. coli and twice as fast as the fastest E. coli.Clearly, a trait found in some Pseudomonas strains allows a significantimprovement in acetate consumption compared to E. coli, and can be usedto enhance the acetate selective cell used in the method of theinvention.

Two general approaches can be used to capitalize on our observations ofthe high growth rate of Pseudomonas on acetate. One approach is to findthe genes encoding the enzymes in Pseudomonas which are responsible forhigh growth rate and transfer them into an E. coli designed to consumeacetate exclusively. To that end, additional Pseudomonas strains,including those that have been completely sequenced, will be evaluatedfor their ability to grow using acetate as a sole carbon source, and thefastest grower will be selected. Genomic DNA will be prepared and largefragments will be cloned into an expression vector to ensure thepreservation of operons. Libraries will be transformed in MG1655, awild-type E. coli strain, and fast growers on a minimal acetate mediumwill be selected. Optionally, the genes responsible for the enhancedgrowth on acetate can then be characterized.

A second approach is to knock out some or all the carbohydrate-consuminggenes in Pseudomonas, so that this strain is becomes acetate-selective.To this end, we can knock out the genes responsible for glucose andxylose consumption in the Pseudomonas strain that has the fastest growthrate on acetate and has been completely sequenced. Because the genesresponsible for glucose and xylose metabolism have been shown to behighly homologous in several bacteria, we will be able to construct therequired knockouts in Pseudomonas.

Essentially, we either bring the Pseudomonas “fast genes” into themicrobe that cannot consume anything else (e.g., an acetate-selective E.coli), or we make a fast-acetate-consumer (e.g., the Pseudomonas strainsthat grow quickly on acetate) unable to consume anything else. For theproduction of biofuels, either Pseudomonas or E. coli can be usedinterchangeably remove the acetate from ligocellulosic hydrolysates. Forthe production of some industrial high-value commodity biochemicals,such as pyruvic acid, where the end use is nutraceuticals, being able toimplement the acetate removal using E. coli may be preferable.

Example 13 Furfural Consumption

We recently isolated about 20 bacterial strains, including strains thatappear visually to be Pseudomonas spp., which consume furfural as thesole carbon source (furfural-selective strains). These strains are idealcandidates for use as inhibitor selective cells in the method of theinvention, where the inhibitor is furfural.

The approach to capitalizing on these cells will be similar to theapproach outlined for developing acetate-selective strains in Example12. Specifically, we will isolate and transfer the gene(s) from theselected isolates which are responsible for its growth furfural into anE. coli strain which has been engineered not to be able to consumesugars. To this end, we will make DNA libraries of genes from selectedisolates, clone them into an E. coli expression vector, transform E.coli and select clones that can grow on furfural. As in Example 12, nospecific genetic information is necessary about the source strain.

Possibly the resulting inhibitor selective strain can be engineered toconsume both acetate and furfural; alternatively, two differentinhibitor selective cells, one that is selective for acetate, andanother that is selective for furfural, can be created and used in themethod of the invention. The acetate-selective strain would consumeacetate exclusively (i.e., it does not have the furfural degradationgenes), and the furfural-selective strain would consume only furfural byhaving an additional knockout in the acetate degradation genes acs andackA.

Example 14 Selective Microbial Removal of Acetate from Sugar Mixtures

Acetic acid is an unavoidable constituent of the biomass hydrolysategenerated from the acetylated hemicellulose and lignin. The removal ofacetate from hydrolysate is necessary for improving the microbialproduction of biochemicals. In this study, acetate is selectivelyremoved from mixtures of glucose and xylose by metabolically engineeredEscherichia coli strain ALS1060 with mutations in the phosphotransferasesystem (PTS) genes of glucose (ptsG, manZ), glucokinase (glk) and xylose(xylA). In batch culture, ALS1060 consumed acetate exclusively at first,and began to consume the two sugars only at a very slow rate when theacetate in the medium was essentially exhausted. In order to eliminateall sugar consumption, we also examined effects of knockouts in the crrgene and glucose non-specific PTS genes malX, fruA, fruB, and bglF inALS1060. The crr knockout showed the least sugar consumption, and abatch process with a strain having five knockouts (ptsG manZ glk xylAcrr) showed less than 1 g/L of sugar consumption after 92 h.

Conversion of lignocellulosic biomass to fuels and chemicals bymicrobial fermentation is a promising alternative to petroleum-basedprocesses (Zaldivar et al., 2001, Appl. Microbiol. Biotechnol. 56:17-34). Lignocellulosic materials are inexpensive and readily available,and are largely carbohydrates in the form of cellulose and hemicellulose(Klinke et al., 2004, Appl. Microbiol. Biotechnol. 66:10-26). However,several challenges remain which limit the wide use of lignocellulosicbiomass. One challenge is that biomass hydrolysates contain inhibitorssuch as acetic acid (acetate). Acetate is an unavoidable product ofhemicellulose depolymerization since xylose is acetylated inlignocellulose (Timmel, 1967, Wood Sci. Technol. 1(1):45-70; Sarkanenand Ludwig, 1971, Lignins: occurrence, formation, structure andreactions. Wiley-Interscience, New York, pp 345-372; Fengel and Wegener,1989, Wood Chemistry, ultrastructure, reactions. Walter de Gruyter,Berlin; Chesson et al., 1993, J. Sci. Food. Agric. 34(12):1330-1340;Torssell, 1997, Natural product chemistry: a mechanistic, biosyntheticand ecological approach. Apotekarsocieteten, Stockholm). Xyloseconversion appears particularly sensitive to acetate, with a 0.15%concentration reducing by 50% the yield of ethanol using E. coli (Nelleet al., 2003, Enzyme. Microb. Technol. 33(6):786-792). Similarly,because its membrane is highly permeable to acetate, S. cerevisiae isparticularly susceptible to acetate inhibition (Casal et al., 1998,Appl. Environ. Microbiol. 64(2):665-668). Acetate also exacerbates otherinhibitory effects; for example, furfuryl alcohol and 2-furfural reduceethanol yield by E. coli more in the presence of acetate (Zaldivar andIngram, 1999, Biotechnol. Bioeng. 66:203-210; Zalivar et al., 1999,Biotechnol. Bioeng. 65:24-33; Zaldivar et al., 2000, Biotechnol. Bioeng.68:524-530). Although the generation of some inhibitors might be reducedby judicious design of the hydrolysis process or by genetic improvementsin the biomass itself, elimination of all acetate in a lignocellulosichydrolysate does not currently seem feasible.

A wide variety of strategies have been proposed to ameliorate the effectof acetate on fermentation (Lasko et al., 2000, Appl. Microbiol.Biotechnol. 54(2):243-247). For example, ion exchange (Horvath et al.,2004, Appl. Biochem. Biotechnol. 114:525-538; Chandel et al., 2007,Biores. Technol. 98:1947-1950) or activated carbon (Benson et al., 2005,Appl. Biochem. Biotechnol. 124:923-934) can prepare reduce acetateconcentration. Similarly, extraction with ethyl acetate reduces aceticacid (and furfural, vanillin and 4-hydroxybenzoic acid), leading to a93% improvement in ethanol yield using Pichia stipitis (Wilson et al.,1989, Appl. Microbiol. Biotechnol. 31:592-596). These approaches involvean additional processing step which significantly affects overallprocess costs (Von Sivers et al., 1994, Biotechnol. Prog. 10:555-560).Preferably, an approach should not only leave the productionmicroorganisms unaffected but remove acetate completely at very lowcost.

We have previously reported a biological strategy for selectivelyremoving components from a mixture (Eiteman et al., 2008, J Biol Eng2:3). The approach involves the “design” of a single strain that willutilize only one component in a mixture. Since many organisms includingEscherichia coli readily consume acetate when this compound is the solecarbon source (Holms 1986, Curr. Top Cell. Regul. 28:69-105), acetatemight be removed from a mixture of xylose, glucose and acetate (forexample) with a strain that is genetically prevented from consumingxylose and glucose. Glucose uptake primarily is mediated byglucosephosphotransferase [ptsG (gpt) encodes the EIICB^(glc) component(Postma et al., 1993, Microbiol. Rev. 57(3):543-594)],mannosephosphotransferase (EC 2.7.1.69) [manZ (mpt) encodes the EIIenzyme (Curtis et al., 1975, J. Bacteriol. 122(3):1189-1199)], andATP-dependent glucokinase (EC 2.7.1.2) encoded by the glk gene (Curtiset al., 1975, J. Bacteriol. 122(3):1189-1199). Knocking out the ptsG,manZ and glk genes prevents E. coli from consuming glucose in a shortbatch process, while a xylA mutant is unable to consume xylose (Eitemanet al., 2008, J Biol Eng 2:3). Thus, a strain with the four knockouts(i.e., ptsG manZ glk xylA) might therefore prevent consumption of bothsugars but allow normal acetate metabolism.

Other cellular processes could also be involved in glucose transport.Due to considerable sequence homology between EII proteins, genescorresponding to other carbohydrate PTS systems might also transferglucose into the cell. For example, the malX gene of the maltose PTSsystem encodes a protein that binds to glucose and displays nearly 35%sequence identity to the protein encoded by ptsG (Reidl and Boos, 1991,J. Bacteriol. 173:4862-4876), and the EII^(fru) protein of the fructosespecific PTS system (expressed by the fruA gene) shows similarity withEllglu (Prior and Kornberg, 1988, J. Gen. Microbiol. 134:2757-2768). Thebgl operon in E. coli (consisting of 3 structural genes bglC, bglS, andbglB) encode components of the specific transport proteinphospho-β-glucosidase (EII^(bgl)) (Schnetz et al., 1987, J.Bacterio1.169:2579-2590) and shows significant sequence homology to thecarboxyl-terminal section of the EII^(glu) protein (Bramley andKornberg, 1987, Proc. Natl. Acad. Sci. USA 84:4777-4780). EIIIAglu isphosphorylated during PEP-phosphotransfer of glucose, and strainslacking the crr gene express nomial levels of all PTS proteins exceptfor EIIA^(glu) (Saier and Roseman, 1976, J Biol. Chem. 251:6598-605).Since none of these carbohydrate transport genes is known to be involvedin acetate metabolism, knockouts of these genes could eliminate anyresidual glucose uptake without affecting acetate consumption.

Using a mixture of xylose, glucose and acetate as a model for biomasshydrolysate, we constructed an acetate-selective strain which does notconsume xylose and glucose, and to demonstrate that this strain couldeffectively remove acetate from a mixture containing these sugars.

Materials and Methods Bacterial Strains

The Escherichia coli strains studied are shown in the following table.

Strain Genotype MG1655 F−λ-rph-1 (wild type) ALS1060 MG1655ΔptsG763::(FRT) ΔmanZ743::(FRT) Δglk-726::(FRT) ΔxylA748::(FRT) ALS1072MG1655 ΔptsG763::(FRT) ΔmanZ743::(FRT) Δglk-726::(FRT) ΔxylA748::KanALS1122 ALS1060 Δcrr::(FRT) ALS1123 ALS1060 ΔfruA::(FRT) ALS1124 ALS1060ΔfruB::(FRT) ALS1125 ALS1060 ΔbglF::(FRT) ALS1127 MalX ALS1122-Kan Thisis the strain with crr knockout and Kan cassette

Shake Flask Growth Conditions

Basal medium (BA10) contained (per L): 13.3 g KH₂PO₄, 4.0 g (NH₄)₂HPO₄,1.2 g MgSO₄.7H₂O, 13.0 mg Zn(CH₃COO)₂.2H₂O, 1.5 mg CuCl₂.2H₂O, 15.0 mgMnCl₂.4H₂O, 2.5 mg CoCl₂.6H₂O, 3.0 mg H₃BO₃, 2.5 mg Na₂MoO₄.2H₂O, 100 mgFe(III)citrate, 8.4 mg Na₂EDTA.2H₂O, 1.7 g citric acid, 0.0045 gthiamine.HCl, and 10 g/L acetate using Na(CH₃COO).3H₂O. BA2 medium wasidentical except it contained 2 g/L acetate. Both BA2 and BA10 mediawere supplemented with xylose and/or glucose as described in the text.Acetate, xylose and glucose were autoclaved separately, sterilycombined, and neutralized with NaOH to a pH of 7.0. Concentrations arereported as acetate without consideration of the counterion.

Growth characteristics of E. coli strains in BA2 medium with 2 g/Lglucose are shown in the following table.

ΔOD/Δt ΔG/Δt Name of strain (AU/h) (mg/L · h) ALS1060 0.018 18.0 ALS1122−0.003 8.4 ALS1123 0.017 16.9 ALS1124 0.016 20.3 ALS1125 0.035 28.7ALS1127 (malX) 0.023 21.4

Growth Conditions

For shake flask experiments, 50 mL BA2 medium contained 2 g/L glucose in250 mL baffled shake flasks at 37° C. and 350 rpm (19 mm pitch).

For fermentation experiments, the selected strain was first grown (5 mL)in a 10 mL shaking test tube containing 5 g/L tryptone, 2.5 g/L yeastextract, 5 g/L NaCl and 2.5 g/L acetate, then transferred to a baffled250 mL shake flask containing 50 mL BA10 incubated at 37° C. and 250 rpm(19 mm pitch). When the OD of the culture reached 2.0-2.5, the contentsof the shake flask were transferred to a bioreactor.

Batch fermentations were carried out at 1.0 L using BA10 in a 2.5 Lbioreactor (Bioflo 2000, New Brunswick Scientific Co. Edison, N.J.,USA). Air was sparged into the fermenter at a flowrate of 1.0 L/min, andthe agitation was 500 rpm to ensure no oxygen limitation. The pH wascontrolled at 7.0 using 20% (w/v) NaOH or 20% (v/v) H₂SO₄, and thetemperature was controlled at 37° C.

Fed-batch experiments initially operated in batch mode and containedBA10 medium but with 5 g/L acetate. When the OD reached 3.0-3.5, sugarsupplemented BA10 medium was fed at an exponentially increasing rate toachieve a constant growth rate of 0.07 h⁻¹. Concentrated NH₄OH was usedfor base control, and the feed solution contained 100 mg/L kanamycin.

Assays

The optical density measured at 600 nm absorbance (OD) (UV-650spectrophotometer, Beckman Instruments, San Jose, Calif.) was used tomonitor cell growth. Glucose, xylose, acetate, and other organicby-products were quantified as previously described (Eiteman andChastain, 1997, Anal. Chim. Acta. 338:69-75).

Results Batch Growth on Acetate

Escherichia coli MG1655 is a common wild-type strain (Jensen 1993, J.Bacteriol. 175:3401-3407) which should consume acetate as the solecarbon source. We first sought to verify this expectation in aerobicbatch growth, and FIG. 15 shows the results in BA10 medium (i.e.,initial concentration of 10 g/L acetate). MG1655 foimed approximately2.5 g/L cells (OD=7.1) at a specific growth rate of 0.23 h⁻¹.

E. coli ALS1060 (see Example 2) has four knockouts of genes coding forproteins involved in the utilization of xylose and glucose: ptsG encodesthe Enzyme IICB^(Glc) of the phosphotransferase system (PTS) forcarbohydrate transport (Postma et al., 1993, Microbiol. Rev.57(3):543-594), manZ encodes the IID^(Man) domain of the mannose PTSpermease (Huber, 1996, Eur. J. Biochem. 239(3):810-817), glk encodesglucokinase (Curtis and Epstein 1975, J. Bacteriol. 122(3):1189-1199)while xylA encodes xylose isomerase. These four mutations should preventthe utilization of either xylose or glucose by ALS1060. In order todetermine whether these mutations had any effect on the growth onacetate, we similarly grew ALS1060 in the same medium (FIG. 15). LikeMG1655, ALS1060 formed 2.5 g/L cells (OD=7.7), and attained a specificgrowth rate of 0.22 h⁻¹.

Extended Batch Growth on Acetate in the Presence of Sugars

Our next objective was to determine whether acetate could be exclusivelyconsumed from a mixture of sugars. Since ALS1060 contains knockoutsinvolved in the consumption of xylose or glucose, the growth of thisstrain in a medium containing xylose, glucose and acetate is expected toidentical to growth in the medium containing acetate alone. In order totest this prediction, we grew ALS1060 in batch culture over an extendedperiod of time in BA10 medium in the presence of either 20 g/L glucose,10 g/L xylose or in a mixture of 10 g/L xylose and 20 g/L glucose

Batch culture using ALS1060 in BA10 medium containing 20 g/L glucose didresult in exclusive acetate consumption during the first 20 h of theprocess (FIG. 16). Moreover, during growth on acetate the specificgrowth rate was 0.21 h⁻¹, identical to the growth rate observed inmedium without acetate (FIG. 15). Interestingly, slow glucoseconsumption commenced about the time acetate was nearly exhausted (FIG.16), and the specific growth rate of ALS1060 after acetate was exhaustedwas found to be 0.014 h⁻¹.

During batch culture using BA10 medium containing 10 g/L xylose ALS1060consumed exclusively acetate, and the concentration of xylose remainedunaltered throughout the process (FIG. 17).

In order to study how the presence of both sugars influenced acetateutilization, ALS1060 was inoculated into BA10 medium with 20 g/L glucoseand 10 g/L xylose. In this case, ALS1060 consumed the acetate in 30 h,during which time less than 2 g/L glucose and 0.6 g/L xylose wasconsumed. Over the next 40 h, however, 7 g/L glucose and 3 g/L xylosewere slowly consumed (FIG. 18).

The extended batch fermentations confirmed that ALS1060 could consumeglucose and xylose in the absence of acetate. Further more it becameclear that the inability of ALS1060 to consume xylose in the absence ofglucose indicates that glucose is necessary for the consumption ofxylose.

Comparison of Knockouts to Prevent Glucose Consumption

Because ALS1060 grew in the presence of glucose albeit slowly, E. colimust have another means to transport and utilize glucose. Mutations inthe ptsG, manZ, and glk genes are insufficient to prevent glucoseconsumption. With the goal of completely eliminating glucoseconsumption, we next examined growth on BA2 with 2 g/L glucose ofstrains having mutations additionally in one of several other geneswhich encoding non-specific PTS proteins: ma/X encoding a protein of themaltose-specific PTS (ALS1127MalX), fruA or fruB encoding proteins ofthe fructose-specific PTS (ALS1123 and ALS1124, respectively); the bgloperon involved in the PTS of β-glucosides (ALS1125), and crr whichencodes the EIIA^(glu) (ALS1122). To compare growth and glucoseconsumption, two parameters were measured: the rate of glucose uptakefor approximately 30 h beyond the time that acetate was exhausted(ΔG/Δt), and the change in the optical density for approximately 30 hbeyond the time that acetate was exhausted (ΔOD/ΔT). Compared toALS1060, only ALS1122 (with the crr mutation) showed a significantlyreduced rate of glucose consumption (8.4 mg/L·h). This strain alsoshowed essentially no change in the optical density over the 30 hperiod, in contrast to the other strains.

Extended Batch Growth of ALS1122-Kan on Acetate in the Presence ofSugars

We next grew the strain containing the five knockouts (ptsG manZ glkxylA crr) and which additionally had drug resistance to kanamycin(“ALS1122-Kan”) in BA10 medium with 20 g/L glucose and 10 g/L xylose.The results are shown in FIG. 19. In this case, less than 1 g/L glucoseor xylose was consumed even after 92 h, even though the acetate had beencompletely consumed after 45 h.

Example 15 Arabinose-Selective and Xylose-Selective W Strains

E. coli is able to use each of the five principal monosaccharides foundin lignocellulosic hydrolysate. We examined the wild-type W strain oneach of these five carbon sources, and the specific growth rates werefound to be 1.00 h⁻¹ (glucose), 0.84 h⁻¹ (xylose), 0.94 h⁻¹ (arabinose),0.66 h⁻¹ (galactose) and 0.46 h⁻¹ (mannose). We selected W to generatesubstrate-selective strains because this strain generally grew fasteston these five carbohydrates.

KD777 is wild-type W strain of E. coli with knockouts in the ptsG, manZand glk genes, a set of genes which has been described as preventinggrowth on glucose (Curtis and Epstein, 1975 J Bacteria 122:1189). In acontrolled batch process KD777 consumed 7 g/L xylose completely in 5.5 hwith a maximum specific growth rate of 0.81 h⁻¹, essentially the samegrowth rate observed when W was grown in this medium. In the presence ofboth xylose and glucose, xylose was again completely consumed in about5.5 h, during which time only 0.3 g/L glucose was consumed (FIG. 20). Inthe presence of glucose the maximum growth rate during xyloseconsumption was 0.78 h⁻¹.

In a controlled batch process KD777 consumed 7 g/L arabinose completelyin less than 5 h with a maximum specific growth rate of 0.84 h⁻¹. In amedium containing both arabinose and glucose, arabinose was consumed in5 h at a mean growth rate of 0.81 h⁻¹, and glucose was not consumedduring the first 14 h of the process (FIG. 21). Thus, arabinose has beenconsumed without any consumption of glucose, and additional mutationswould permit this strain to accumulate a desired product from arabinosewithout affecting glucose.

Additional crr Knockout

KD915 contains the ptsG, glk and manZ knockouts like KD777, butadditionally has a knockout in the crr gene. The crr gene encodes forthe Enzyme IIA component of the phosphotransferase system for glucoseuptake. When KD915 consumed xylose as the sole carbon source, thisstrain attained a specific growth rate of 0.65 h⁻¹, about 20% lower thanobserved for KD777. In the presence of both xylose and glucose, xylosewas consumed at a growth rate of 0.62 h⁻¹ (FIG. 22). Similarly, 7 g/Larabinose as the sole carbon source was consumed by KD915 with aspecific growth rate of 0.64 h⁻¹, about 20% lower than KD777. In amedium containing both arabinose and glucose, arabinose was consumed ata mean growth rate of 0.64 h⁻¹, and glucose was consumed only slowlyonly after arabinose was depleted (FIG. 23).

Example 16 Ethanol Production in Sugar Mixtures

ALS1074 (MG1655 xylA pflB) contains a knockout of the xylA gene andtherefore is expected in a mixture of glucose and xylose to beglucose-selective; i.e., the strain will consume glucose but not xylose.ALS1073 (MG1655 ptsG manZ glk pflB) contains three knockouts in glucoseuptake and is therefore expected in a mixture of glucose and xylose tobe xylose-selective; i.e., the strain will consume xylose but notglucose. These two strains were transformed with plasmidpTrc99A-pdc.adhB containing the pyruvate decarboxylase (pdc) and alcoholdehydrogenase (adhB) genes from Zymomonas mobilis. The presence of thesetwo enzymes has been previously shown to endow E. coli with the abilityto accumulate significant ethanol (Ingram and Conway 1988 Appl. Env.Microbiol. 54(2):397-404). ALS1073 and ALS1074 were individually andsimultaneously grown in a medium containing nominally 15 g/L glucose and10 g/L xylose. Specifically, 50 mL of medium was used in a 250 mL shakeflask operating at a temperature of 37 C and an agitation of 250 rpm.The cultures were induced with 1 mM IPTG 8 h after inoculation.Anaerobic conditions were simulated 12 h after inoculation by reducingthe agitation to 75 rpm. For each of the three experiments performed induplicate (ALS1073 alone, ALS1074 alone, and ALS1073 and ALS1074together), a sample was taken at the beginning of the experiment, andagain at the end. The results are shown in the following table.

ALS1073 + ALS1074 alone ALS1073 alone ALS1074 Exp 1 Exp 2 Exp 1 Exp 2Exp 1 Exp 2 Xylose (g/L) Start 9.8 10.0 9.2 8.9 8.6 8.8 End 9.7 9.9 2.21.5 0.1 0 Glucose (g/L) Start 13.1 13.5 14.0 13.6 12.8 13.1 End 0.4 0.213.3 13.5 0.3 0.2 Ethanol (g/L) 2.8 2.9 2.2 2.4 5.7 5.8 Optical Density9.0 7.6 2.0 2.2 8.7 9.1

For ALS1074, essentially all of the glucose was consumed and none of thexylose, resulting in an accumulation of about 2.8 g/L ethanol. ForALS1073, essentially all of the xylose was consumed but none of theglucose, resulting in an accumulation of an average of 2.3 g/L ethanol.When both ALS1073 and ALS1074 were used together on this sugar mixture,both sugars were consumed with about 5.7 g/L ethanol generated. Theresults demonstrate that the two-strain approach, in which each strainis designed to be selective for one sugar but the strains otherwise areidentical, can effectively generate ethanol.

Example 17 Batch Growth of KD777 with Additional Pentose

Results with batch growth of KD777 in xylose-glucose orarabinose-glucose mixtures suggested that the onset of glucoseconsumption did not occur until the pentose was depleted. We sought toverify whether the presence of pentose effectively hindered glucosemetabolism in this strain by providing the culture with additionalpentose after the initial quantity of pentose had been exhausted. Forthe case of a xylose-glucose mixture, we repeated the previousexperiment, but added xylose to a concentration of about 7 g/L six hoursafter xylose was depleted, when the glucose concentration wasapproximately half its initial concentration (FIG. 7). The additionalxylose was consumed within 3 h, and during the initial part of thatinterval the glucose concentration remained fixed. At the time when thexylose was just being depleted the glucose concentration increased fromabout 3 g/L to 4 g/L. After that additional dose of xylose had beenexhausted, glucose consumption resumed and at a rate slower than therate observed prior to the xylose addition.

For the case of an arabinose-glucose mixture, approximately 7 g/Larabinose was added about 20 h after arabinose was depleted, when theglucose concentration had reached approximately half its initialconcentration (FIG. 8). This additional arabinose was consumed in twohours, and during that time the glucose concentration at first remainedthe same and then increased from about 3.5 g/L to 4.2 g/L. After thatadditional does of arabinose had been exhausted, glucose consumptionresumed at approximately the same rate observed prior to the arabinoseaddition.

Without intending to be bound by theory, these observations may indicatethat glucose is leaking into the cell through the xylosetransport/metabolic pathway, and that it may be difficult in theseparticular cells to eliminate glucose uptake completely. Operationally,this is not a problem if fed-batch processing is used. If a continuousculture was run, it may be advantageous to keep the xylose concentrationhigher than a threshold value at which glucose begins to leak throughthe glucose-deficient strains. It may be prudent to maintain xylosemetabolism in the continuous process, as opposed to a batch process,because over the course of a long time interval, the population of thecultures in the continuous culture could otherwise change in asuboptimal way.

Example 18 Sugar-Selective Cells Engineered to Accumulate Pyruvate

Earlier we demonstrated that 1.0 M (90 g/L) pyruvic acid (henceforthcalled pyruvate) can accumulate in less than 40 h with 70% mass yieldfrom glucose in E. coli containing knockouts in the aceEF, pps, ldhA,poxB, pflB, arcA, and atpFH genes (Zhu et al., 2008. Appl. Environ.Microbia 74:6649). Pyruvate is a key precursor to numerous biochemicalproducts including isobutanol (Atsumi et al., 2008. Nature 451:84-90),and in order to enhance accumulation of this precursor, one or more ofthese additional mutations can be introduced into in varioussugar-specific strains of E. coli. The optimal xylose-to-pyruvateconstruct may not be identical to the optimal glucose-to-pyruvateconstruct, because the two mutations that reduce the biomass generationfrom glucose (arcA and atpFH) may be detrimental when pentoses such asxylose are the source for pyruvate production, since pentoses do notprovide the cell with as much ATP as glucose. Arabinose- andxylose-specific strains which lack the arcA and atpFH mutations can alsobe constructed.

Example 19 Sugar-Selective Cells Engineered to Accumulate Succinate

Succinic acid (henceforth called succinate) and its derivatives arewidely used as specialty chemicals in foods, pharmaceuticals, andcosmetics (Guettler et al. 1998), and it can serve as a startingmaterial for many commercially important products (Zeikus et al. 1999).Moreover, succinate is listed among the U.S. Dept. of Energy's top 30commodity chemicals from biomass (U.S. DOE 2004). During the anaerobicproduction of succinate, organisms fix the greenhouse gas CO₂ viacarboxylation reactions and convert C₃ to C₄ metabolites. Throughprevious work, we have demonstrated that 0.8 M succinate (100 g/L) canaccumulate in 72 h with 110% mass yield from glucose in E. colicontaining knockouts in the pflB, ldhA and ptsG genes and harboring thepyruvate carboxylase (pyc) gene (see U.S. Pat. No. 7,749,740; Gokarn etal., Appl. Microbiol. Biotechnol., 56, 188-195 (2001); and Vemuri etal., Appl. Environ. Microbiol. 68(4):1715-27 (2002)).

A process for generating succinate is quite different than a process forpyruvate, and that has important consequences for thesubstrate-selective approach. Pyruvate is a growth-associated product,and thus the focus of the process is to insure that the individualstrains are growing on their respective substrate. The process togenerate succinate involves its accumulation as a non-growth associatedproduct, and therefore the focus is insuring that the production phaseis aligned so that each of the substrates are converted into the productsuccinate in proportion to each carbohydrate's availability. See Vemuriet al., J. Ind. Microbiol. Biotechnol., 28(6), 325-332 (2002); Vemuri etal., Appl. Env. Microbiol., 68(4), 1715-1727 (2002); and Challener,Specialty Chemicals Magazine 6:42-44 (2009)).

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference.

The foregoing detailed description and examples have been given forclarity of understanding only. No unnecessary limitations are to beunderstood therefrom. The invention is not limited to the exact detailsshown and described, for variations obvious to one skilled in the artwill be included within the invention defined by the claims.

1. A method for producing a biochemical comprising: contacting anorganic material comprising a mixture of sugars, with a plurality ofsugar-selective cells comprising at least first and secondsugar-selective cells, wherein a first sugar-selective cell selectivelymetabolizes a first sugar, and a second sugar-selective cell selectivelymetabolizes a second sugar, to yield a co-culture of sugar-selectivecells, under conditions to allow the plurality of sugar-selective cellsto produce the biochemical; assaying the co-culture periodically for thepresence or amount of at least one of the first or second sugars; andadjusting at least one parameter selected from the group consisting of:the amount of at least one sugar-selective cell in the co-culture; theactivity of at least one sugar-selective cell in the co-culture; and thetiming of the contact of at least one sugar-selective cell with theorganic material; so as to increase the rate of consumption of at leastone of the sugars.
 2. The method of claim 1 wherein the plurality ofsugar-selective cells comprises at least one hexose-selective cell andat least one pentose-selective cell.
 3. The method of claim 2 whereinthe hexose-selective cell comprises a glucose-selective cell, andwherein the pentose-selective cell comprises a xylose-selective cell. 4.The method of claim 1 wherein at least one of the plurality ofsugar-selective cells cannot metabolize the first sugar, and at leastone of the plurality of sugar-selective cells cannot metabolize thesecond sugar, and wherein the first and second sugars are independentlyselected from the group consisting of glucose, xylose, arabinose,galactose, and mannose.
 5. The method of claim 1 wherein at least one ofthe plurality of sugar-selective cells cannot metabolize at least twosugars independently selected from the group consisting of glucose,xylose, arabinose, galactose, and mannose.
 6. The method of claim 1wherein at least one of the plurality of sugar-selective cells cannotmetabolize at least three sugars independently selected from the groupconsisting of glucose, xylose, arabinose, galactose, and mannose.
 7. Themethod of claim 1 wherein at least one of the plurality ofsugar-selective cells can metabolize only one sugar independentlyselected from the group consisting of glucose, xylose, arabinose,galactose, and mannose.
 8. The method of claim 1 wherein each of theplurality of sugar-selective cells is independently selected from thegroup consisting of Escherichia coli, Zymomonas mobilis, Corynebacteriumglutamicum, Lactic Acid Bacteria, Saccharomyces cerevisiae, Pichiastipitis and Ambrosiozyma monospora.
 9. The method of claim 1 whereineach of the plurality of sugar-selective cells is a member of the samespecies.
 10. The method of claim 9 wherein the species is a bacterialspecies or a yeast species.
 11. The method of claim 10 wherein thebacterial species is E. coli.
 12. The method of claim 1 wherein theplurality of sugar-selective cells comprises: a first E. coli cellcomprising a modification or deletion of at least one gene involved inglucose metabolism so as to inhibit or prevent the first E. coli cellfrom consuming glucose; and a second E. coli cell comprising amodification or deletion of at least one gene involved in xylosemetabolism so as to inhibit or prevent the second E. coli cell fromconsuming xylose.
 13. The method of claim 1 wherein the biochemical isethanol, and wherein at least one of the plurality of sugar-selectivecells has been genetically engineered for enhanced ethanol production.14. The method of claim 13 wherein at least one of plurality ofsugar-selective cells has been genetically engineered to express oroverexpress an alcohol dehydrogenase enzyme and/or a pyruvatedecarboxylase enzyme.
 15. The method of claim 1 wherein the biochemicalis selected from the group consisting of ethanol, butanol, succinate,lactate, fumarate, pyruvate, butyric acid and acetone.
 16. The method ofclaim 1 further comprising contacting the organic material with at leastone inhibitor-selective cell selective for an inhibitor selected fromthe group consisting of acetic acid, furfural and hydroxymethyl furfural(HMF) under conditions to allow the inhibitor-selective cell tometabolize the inhibitor.
 17. The method of claim 16 wherein theinhibitor-selective cell comprises a modification or deletion of the crrgene.
 18. The method of claim 16 wherein the inhibitor-selective cell isan E. coli cell comprising a modification or deletion of at least onegene involved in glucose metabolism so as to inhibit or prevent the cellfrom consuming glucose, and a modification or deletion of at least onegene involved in xylose metabolism so as to inhibit or prevent the cellfrom consuming xylose.
 19. The method of claim 16 wherein theinhibitor-selective cell has been genetically engineered such that itcannot metabolize any sugar selected from the group consisting ofglucose, xylose, arabinose, galactose and mannose.
 20. The method ofclaim 16 wherein the inhibitor-selective cell is contacted with theorganic material under conditions to allow the inhibitor-selective cellto produce the biochemical.
 21. The method of claim 16 whereincontacting the organic material with at least one inhibitor-selectivecell occurs prior to contacting the organic material with the pluralityof sugar-selective cells.
 22. The method of claim 16 wherein contactingthe organic material with at least one inhibitor-selective cell occursconcurrent with contacting the organic material with the plurality ofsugar-selective cells.
 23. The method of claim 1 further comprisinghydrolyzing a lignocellulosic biomass to produce the organic material.24. The method of claim 1 further comprising purifying the biochemical.25-56. (canceled)
 57. A method for producing pyruvate comprising:concurrently contacting an organic material comprising a mixture ofsugars with a plurality of sugar-selective cells under conditions toallow the plurality of sugar-selective cells to produce pyruvate,wherein at least one sugar-selective cell is a bacterial cell containingknockouts of a plurality of genes selected from the group consisting ofaceEF, pps, ldhA, poxB, pflB, arcA, and atpFH, or any combinationthereof.
 58. The method of claim 57 wherein the bacterial cell comprisesa pentose-selective bacterial cell containing knockouts of a pluralityof genes selected from the group consisting of aceEF, pps, ldhA, poxB,and pflB, or any combination thereof. 59-60. (canceled)
 61. The methodof claim 57 wherein the plurality of sugar-selective cells comprises atleast one hexose-selective cell and at least one pentose-selective cell.62. The method of claim 57 wherein the plurality of sugar-selectivecells comprises first and second sugar-selective cells, wherein thefirst sugar-selective cell metabolizes a first sugar that cannot bemetabolized by the second sugar-selective cell, and the secondsugar-selective cell metabolizes a second sugar that cannot bemetabolized by the first sugar-selective cell, and wherein the first andsecond sugars are independently selected from the group consisting ofglucose, xylose, arabinose, galactose, and mannose.
 63. The method ofclaim 57 wherein at least one of the plurality of sugar-selective cellscannot metabolize at least two sugars independently selected from thegroup consisting of glucose, xylose, arabinose, galactose, and mannose.64. The method of claim 57 wherein the bacterial cell is E. coli.